Polynucleotide molecule from haematococcus pluvialis encoding a polypeptide having a ⊕-c-4-oxygenase activity for biotechnological production of (3s,3&#39;s) astaxanthin and its specific expression in chromoplasts of higher plants

ABSTRACT

The present invention relates, in general, to a biotechnological method for production of (3S,3′S) astaxanthin. In particular, the present invention relates to a peptide having a β-C-4-oxygenase activity; a DNA segment coding for this peptide; an RNA segments coding for this peptide; a recombinant DNA molecule comprising a vector and the DNA segment; a host cell or organism containing the above described recombinant DNA molecule or DNA segment; and to a method of biotechnologically producing (3S,3′S) astaxanthin or a food additive containing (3S,3′S) astaxanthin, using the host.

This is a continuation of U.S. patent application Ser. No. 09/259,294,filed Mar. 1, 1999, now U.S. Pat. No. 6,218,599, which is a continuationof U.S. patent application Ser. No. 08/742,605, filed Oct. 28, 1996, nowU.S. Pat. No. 5,965,795, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/562,535, filed Nov. 24, 1995, now U.S. Pat. No.5,916,791, issued Jun. 29, 1999.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates, in general, to a biotechnological methodfor production of (3S,3′S) astaxanthin. In particular, the presentinvention relates to a peptide having a β-C-4-oxygenase activity; a DNAsegment coding for this peptide; an RNA segments coding for thispeptide; a recombinant DNA molecule comprising a vector and the DNAsegment; a host cell or organism containing the above describedrecombinant DNA molecule or DNA segment; and to a method ofbiotechnologically producing (3S,3′S) astaxanthin or a food additivecontaining (3S,3′S) astaxanthin, using the host.

Carotenoids, such as astaxanthin, are natural pigments that areresponsible for many of the yellow, orange and red colors seen in livingorganisms. Carotenoids are widely distributed in nature and have, invarious living systems, two main biological functions: they serve aslight-harvesting pigments in photosynthesis, and they protect againstphotooxidative damage. These and additional biological functions ofcarotenoids, their important industrial role, and their biosynthesis arediscussed hereinbelow.

As part of the light-harvesting antenna, carotenoids can absorb photonsand transfer the energy to chlorophyll, thus assisting in the harvestingof light in the range of 450-570 nm [see, Cogdell R J and Frank H A(1987) How carotenoids function in photosynthestic bacteria. BiochimBiophys Acta 895: 63-79; Cogdell R (1988) The function of pigments inchloroplasts. In: Goodwin T W (ed) Plant Pigments, pp 183-255. AcademicPress, London; Frank H A, Violette C A, Trautman J K, Shreve A P, OwensT G and Albrecht A C (1991) Carotenoids in photosynthesis: structure andphotochemistry. Pure Appl Chem 63: 109-114; Frank H A, Farhoosh R,Decoster B and Christensen R L (1992) Molecular features that controlthe efficiency of carotenoid-to-chlorophyll energy transfer inphotosynthesis. In: Murata N (ed) Research in Photosynthesis, Vol I, pp125-128. Kluwer, Dordrecht; and, Cogdell R J and Gardiner A T (1993)Functions of carotenoids in photosynthesis. Meth Enzymol 214: 185-193].Although carotenoids are integral constituents of the protein-pigmentcomplexes of the light-harvesting antennae in photosynthetic organisms,they are also important components of the photosynthetic reactioncenters.

Most of the total carotenoids is located in the light harvesting complexII [Bassi R, Pineaw B, Dainese P and Marquartt J (1993) Carotenoidbinding proteins of photosystem II. Eur J Biochem 212: 297-302]. Theidentities of the photosynthetically active carotenoproteins and theirprecise location in light-harvesting systems are not known. Carotenoidsin photochemically active chlorophyll-protein complexes of thethermophilic cyanobacterium Synechococcus sp. were investigated bylinear dichroism spectroscopy of oriented samples [see, Breton J andKato S (1987) Orientation of the pigments in photosystem II:low-temperature linear-dichroism study of a core particle and of itschlorophyll-protein subunits isolated from Synechococcus sp. BiochimBiophys Acta 892: 99-107]. These complexes contained mainly a β-carotenepool absorbing around 505 and 470 nm, which is oriented close to themembrane plane. In photochemically inactive chlorophyll-proteincomplexes, the β-carotene absorbs around 495 and 465 nm, and themolecules are oriented perpendicular to the membrane plane.

Evidence that carotenoids are associated with cyanobacterial photosystem(PS) II has been described [see, Suzuki R and Fujita Y (1977) Carotenoidphotobleaching induced by the action of photosynthetic reaction centerII: DCMU sensitivity. Plant Cell Physiol 18: 625-631; and, Newman P Jand Sherman L A (1978) Isolation and characterization of photosystem Iand II membrane particles from the blue-green alga Synechococcuscedrorum. Biochim Biophys Acta 503: 343-361]. There are two β-carotenemolecules in the reaction center core of PS II [see, Ohno T, Satoh K andKatoh S (1986) Chemical composition of purified oxygen-evolvingcomplexes from the thermophilic cyanobacterium Synechococcus sp. BiochimBiophys Acta 852: 1-8; Gounaris K, Chapman D J and Barber J (1989)Isolation and characterization of a D1/D2/ cytochrome b-559 complex fromSynechocystis PCC6803. Biochim Biophys Acta 973: 296-301; and, Newell RW, van Amerongen H, Barber J and van Grondelle R (1993) Spectroscopiccharacterization of the reaction center of photosystem II usingpolarized light: Evidence for β-carotene excitors in PS II reactioncenters. Biochim Biophys Acta 1057: 232-238] whose exact function(s) isstill obscure [reviewed by Satoh K (1992) Structure and function of PSII reaction center. In: Murata N (ed) Research in Photosynthesis, Vol.II, pp. 3-12. Kluwer, Dordrecht]. It was demonstrated that these twocoupled β-carotene molecules protect chlorophyll P680 from photodamagein isolated PS II reaction centers [see, De Las Rivas J, Telfer A andBarber J (1993) 2-coupled β-carotene molecules protect P680 fromphotodamage in isolated PS II reaction centers. Biochim. Biophys. Acta1142: 155-164], and this may be related to the protection againstdegradation of the D1 subunit of PS II [see, Sandmann G (1993) Genes andenzymes involved in the desaturation reactions from phytoene tolycopene. (abstract), 10th International Symposium on Carotenoids,Trondheim CL 1-2]. The light-harvesting pigments of a highly purified,oxygen-evolving PS II complex of the thermophilic cyanobacteriumSynechococcus sp. consists of 50 chlorophyll α and 7 β-carotene, but noxanthophyll, molecules [see, Ohno T, Satoh K and Katoh S (1986) Chemicalcomposition of purified oxygen-evolving complexes from the thermophiliccyanobacterium Synechococcus sp. Biochim Biophys Acta 852: 1-8].β-carotene was shown to play a role in the assembly of an active PS IIin green algae [see, Humbeck K, Romer S and Senger H (1989) Evidence forthe essential role of carotenoids in the assembly of an active PS II.Planta 179: 242-250].

Isolated complexes of PS I from Phormidium luridum, which contained 40chlorophylls per P700, contained an average of 1.3 molecules ofβ-carotene [see, Thornber J P, Alberte R S, Hunter F A, Shiozawa J A andKan K S (1976) The organization of chlorophyll in the plantphotosynthetic unit. Brookhaven Symp Biology 28: 132-148]. In apreparation of PS I particles from Synechococcus sp. strain PCC 6301,which contained 130±5 molecules of antenna chlorophylls per P700, 16molecules of carotenoids were detected [see, Lundell D J, Glazer A N,Melis A and Malkin R (1985) Characterization of a cyanobacterialphotosystem I complex. J Biol Chem 260: 646-654]. A substantial contentof β-carotene and the xanthophylls cryptoxanthin and isocryptoxanthinwere detected in PS I pigment-protein complexes of the thermophiliccyanobacterium Synechococcus elongatus [see, Coufal J, Hladik J andSofrova D (1989) The carotenoid content of photosystem 1 pigment-proteincomplexes of the cyanobacterium Synechococcus elongatus. Photosynthetica23: 603-616]. A subunit protein-complex structure of PS I from thethermophilic cyanobacterium Synechococcus sp., which consisted of fourpolypeptides (of 62, 60, 14 and 10 kDa), contained approximately 10β-carotene molecules per P700 [see, Takahashi Y, Hirota K and Katoh S(1985) Multiple forms of P700-chlorophyll α-protein complexes fromSynechococcus sp.: the iron, quinone and carotenoid contents. PhotosynthRes 6: 183-192]. This carotenoid is exclusively bound to the largepolypeptides which carry the functional and antenna chlorophyll α. Thefluorescence excitation spectrum of these complexes suggested thatβ-carotene serves as an efficient antenna for PS I.

As mentioned, an additional essential function of carotenoids is toprotect against photooxidation processes in the photosynthetic apparatusthat are caused by the excited triplet state of chlorophyll. Carotenoidmolecules with π-electron conjugation of nine or more carbon—carbondouble bonds can absorb triplet-state energy from chlorophyll and thusprevent the formation of harmful singlet-state oxygen radicals. InSynechococcus sp. the triplet state of carotenoids was monitored inclosed PS II centers and its rise kinetics of approximately 25nanoseconds is attributed to energy transfer from chlorophyll tripletsin the antenna [see, Schlodder E and Brettel K (1988) Primary chargeseparation in closed photosystem II with a lifetime of 11 nanoseconds.Flash-absorption spectroscopy with oxygen-evolving photosystem IIcomplexes from Synechococcus. Biochim Biophys Acta 933: 22-34]. It isconceivable that this process, that has a lower yield compared to theyield of radical-pair formation, plays a role in protecting chlorophyllfrom damage due to over-excitation.

The protective role of carotenoids in vivo has been elucidated throughthe use of bleaching herbicides such as norflurazon that inhibitcarotenoid biosynthesis in all organisms performing oxygenicphotosynthesis [reviewed by Sandmann G and Boger P (1989) Inhibition ofcarotenoid biosynthesis by herbicides. In: Boger P and Sandmann G (Eds.)Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton,Fla.]. Treatment with norflurazon in the light results in a decrease ofboth carotenoid and chlorophyll levels, while in the dark, chlorophylllevels are unaffected. Inhibition of photosynthetic efficiency in cellsof Oscillatoria agardhii that were treated with the pyridinoneherbicide, fluridone, was attributed to a decrease in the relativeabundance of myxoxanthophyll, zeaxanthin and β-carotene, which in turncaused photooxidation of chlorophyll molecules [see, Canto de Loura I,Dubacq J P and Thomas J C (1987) The effects of nitrogen deficiency onpigments and lipids of cianobacteria. Plant Physiol 83: 838-843].

It has been demonstrated in plants that zeaxanthin is required todissipate, in a nonradiative manner, the excess excitation energy of theantenna chlorophyll [see, Demmig-Adams B (1990) Carotenoids andphotoprotection in plants: a role for the xanthophyll zeaxanthin.Biochim Biophys Acta 1020: 1-24; and, Demmig-Adams B B and Adams WW III(1990) The carotenoid zeaxanthin and high-energy-state quenching ofchlorophyll fluorescence. Photosynth Res 25: 187-197]. In algae andplants a light-induced deepoxidation of violaxanthin to yieldzeaxanthin, is related to photoprotection processes [reviewed byDemmig-Adams B and Adams WW III (1992) Photoprotection and otherresponses of plants to high light stress. Ann Rev Plant Physiol PlantMol Biol 43: 599-626]. The light-induced deepoxidation of violaxanthinand the reverse reaction that takes place in the dark, are known as the“xanthophyll cycle” [see, Demmig-Adams B and Adams WW III (1992)Photoprotection and other responses of plants to high light stress. AnnRev Plant Physiol Plant Mol Biol 43: 599-626]. Cyanobacterial lichens,that do not contain any zeaxanthin and that probably are incapable ofradiationless energy dissipation, are sensitive to high light intensity;algal lichens that contain zeaxanthin are more resistant to high-lightstress [see, Demmig-Adams B, Adams WW III, Green T G A, Czygan F C andLange O L (1990) Differences in the susceptibility to light stress intwo lichens forming a phycosymbiodeme, one partner possessing and onelacking the xanthophyll cycle. Oecologia 84: 451-456; Demmig-Adams B andAdams WW III (1993) The xanthophyll cycle, protein turnover, and thehigh light tolerance of sun-acclimated leaves. Plant Physiol 103:1413-1420; and, Demmig-Adams B (1990) Carotenoids and photoprotection inplants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta1020: 1-24]. In contrast to algae and plants, cyanobacteria do not havea xanthophyll cycle. However, they do contain ample quantities ofzeaxanthin and other xanthophylls that can support photoprotection ofchlorophyll.

Several other functions have been ascribed to carotenoids. Thepossibility that carotenoids protect against damaging species generatedby near ultra-violet (UV) irradiation is suggested by results describingthe accumulation of β-carotene in a UV-resistant mutant of thecyanobacterium Gloeocapsa alpicola [see, Buckley C E and Houghton J A(1976) A study of the effects of near UV radiation on the pigmentationof the blue-green alga Gloeocapsa alpicola. Arch Microbiol 107: 93-97].This has been demonstrated more elegantly in Escherichia coli cells thatproduce carotenoids [see, Tuveson R W and Sandmann G (1993) Protectionby cloned carotenoid genes expressed in Escherichia coli againstphototoxic molecules activated by near-ultraviolet light. Meth Enzymol214: 323-330]. Due to their ability to quench oxygen radical species,carotenoids are efficient anti-oxidants and thereby protect cells fromoxidative damage. This function of carotenoids is important in virtuallyall organisms [see, Krinsky N I (1989) Antioxidant functions ofcarotenoids. Free Radical Biol Med 7: 617-635; and, Palozza P andKrinsky N I (1992) Antioxidant effects of carotenoids in vivo and invitro—an overview. Meth Enzymol 213: 403-420]. Other cellular functionscould be affected by carotenoids, even if indirectly. Althoughcarotenoids in cyanobacteria are not the major photoreceptors forphototaxis, an influence of carotenoids on phototactic reactions, thathave been observed in Anabaena variabilis, was attributed to the removalof singlet oxygen radicals that may act as signal intermediates in thissystem [see, Nultsch W and Schuchart H (1985) A model of the phototacticreaction chain of cyanobacterium Anabaena variabilis. Arch Microbiol142: 180-184].

In flowers and fruits carotenoids facilitate the attraction ofpollinators and dispersal of seeds. This latter aspect is stronglyassociated with agriculture. The type and degree of pigmentation infruits and flowers are among the most important traits of many crops.This is mainly since the colors of these products often determine theirappeal to the consumers and thus can increase their market worth.

Carotenoids have important commercial uses as coloring agents in thefood industry since they are non-toxic [see, Bauernfeind J C (1981)Carotenoids as colorants and vitamin A precursors. Academic Press,London]. The red color of the tomato fruit is provided by lycopene whichaccumulates during fruit ripening in chromoplasts. Tomato extracts,which contain high content (over 80% dry weight) of lycopene, arecommercially produced worldwide for industrial use as food colorant.Furthermore, the flesh, feathers or eggs of fish and birds assume thecolor of the dietary carotenoid provided, and thus carotenoids arefrequently used in dietary additives for poultry and in aquaculture.Certain cyanobacterial species, for example Spirulina sp. [see, Sommer TR, Potts W T and Morrissy N M (1990) Recent progress in processedmicroalgae in aquaculture. Hydrobiologia 204/205: 435-443], arecultivated in aquaculture for the production of animal and human foodsupplements. Consequently, the content of carotenoids, primarily ofβ-carotene, in these cyanobacteria has a major commercial implication inbiotechnology.

Most carotenoids are composed of a C₄₀ hydrocarbon backbone, constructedfrom eight C₅ isoprenoid units and contain a series of conjugated doublebonds. Carotenes do not contain oxygen atoms and are either linear orcyclized molecules containing one or two end rings. Xanthophylls areoxygenated derivatives of carotenes. Various glycosilated carotenoidsand carotenoid esters have been identified. The C₄₀ backbone can befurther extended to give C₄₅ or C₅₀ carotenoids, or shortened yieldingapocarotenoids. Some nonphotosynthetic bacteria also synthesize C₃₀carotenoids. General background on carotenoids can be found in Goodwin TW (1980) The Biochemistry of the Carotenoids, Vol. 1, 2nd Ed. Chapmanand Hall, New York; and in Goodwin T W and Britton G (1988) Distributionand analysis of carotenoids. In: Goodwin T W (ed) Plant Pigments, pp62-132. Academic Press, New York.

More than 640 different naturally-occurring carotenoids have been so farcharacterized, hence, carotenoids are responsible for most of thevarious shades of yellow, orange and red found in microorganisms, fungi,algae, plants and animals. Carotenoids are synthesized by allphotosynthetic organisms as well as several nonphotosynthetic bacteriaand fungi, however they are also widely distributed through feedingthroughout the animal kingdom.

Carotenoids are synthesized de novo from isoprenoid precursors only inphotosynthetic organisms and some microorganisms, they typicallyaccumulate in protein complexes in the photosynthetic membrane, in thecell membrane and in the cell wall.

As detailed in FIG. 1, in the biosynthesis pathway of β-carotene, fourenzymes convert geranylgeranyl pyrophosphate of the central isoprenoidpathway to β-carotene. Carotenoids are produced from the generalisoprenoid biosynthetic pathway. While this pathway has been known forseveral decades, only recently, and mainly through the use of geneticsand molecular biology, have some of the molecular mechanisms involved incarotenoids biogenesis, been elucidated. This is due to the fact thatmost of the enzymes which take part in the conversion of phytoene tocarotenes and xanthophylls are labile, membrane-associated proteins thatlose activity upon solubilization [see, Beyer P, Weiss G and Kleinig H(1985) Solubilization and reconstitution of the membrane-boundcarotenogenic enzymes from daffodils chromoplasts. Eur J Biochem 153:341-346; and, Bramley P M (1985) The in vitro biosynthesis ofcarotenoids. Adv Lipid Res 21: 243-279]. However, solubilization ofcarotenogenic enzymes from Synechocystis sp. strain PCC 6714 that retainpartial activity has been reported [see, Bramley P M and Sandmann G(1987) Solubilization of carotenogenic enzyme of Aphanocapsa. Phytochem26: 1935-1939]. There is no genuine in vitro system for carotenoidbiosynthesis which enables a direct essay of enzymatic activities. Acell-free carotenogenic system has been developed [see, Clarke I E,Sandmann G, Bramley P M and Boger P (1982) Carotene biosynthesis withisolated photosynthetic membranes. FEBS Lett 140: 203-206] and adaptedfor cyanobacteria [see, Sandmann G and Bramley P M (1985) Carotenoidbiosynthesis by Aphanocapsa homogenates coupled to a phytoene-generatingsystem from Phycomyces blakesleeanus. Planta 164: 259-263; and, BramleyP M and Sandmann G (1985) In vitro and in vivo biosynthesis ofxanthophylls by the cyanobacterium Aphanocapsa. Phytochem 24:2919-2922]. Reconstitution of phytoene desaturase from Synechococcus sp.strain PCC 7942 in liposomes was achieved following purification of thepolypeptide, that had been expressed in Escherichia coli [see, Fraser PD, Linden H and Sandmann G (1993) Purification and reactivation ofrecombinant Synechococcus phytoene desaturase from an overexpressingstrain of Escherichia coli. Biochem J 291: 687-692].

Referring now to FIG. 1, carotenoids are synthesized from isoprenoidprecursors. The central pathway of isoprenoid biosynthesis may be viewedas beginning with the conversion of acetyl-CoA to mevalonic acid.D³-isopentenyl pyrophosphate (IPP), a C₅ molecule, is formed frommevalonate and is the building block for all long-chain isoprenoids.Following isomerization of IPP to dimethylallyl pyrophosphate (DMAPP),three additional molecules of IPP are combined to yield the C₂₀molecule, geranylgeranyl pyrophosphate (GGPP). These 1′-4 condensationreactions are catalyzed by prenyl transferases [see, Kleinig H (1989)The role of plastids in isoprenoid biosynthesis. Ann Rev Plant PhysiolPlant Mol Biol 40: 39-59]. There is evidence in plants that the sameenzyme, GGPP synthase, carries out all the reactions from DMAPP to GGPP[see, Dogbo O and Camara B (1987) Purification of isopentenylpyrophosphate isomerase and geranylgeranyl pyrophosphate synthase fromCapsicum chromoplasts by affinity chromatography. Biochim Biophys Acta920: 140-148; and, Laferriere A and Beyer P (1991) Purification ofgeranylgeranyl diphosphate synthase from Sinapis alba etioplasts.Biochim Biophys Acta 216: 156-163].

The first step that is specific for carotenoid biosynthesis is thehead-to-head condensation of two molecules of GGPP to produceprephytoene pyrophosphate (PPPP). Following removal of thepyrophosphate, GGPP is converted to 15-cis-phytoene, a colorless C₄₀hydrocarbon molecule. This two-step reaction is catalyzed by the solubleenzyme, phytoene synthase, an enzyme encoded by a single gene (crtB), inboth cyanobacteria and plants [see, Chamovitz D, Misawa N, Sandmann Gand Hirschberg J (1992) Molecular cloning and expression in Escherichiacoli of a cyanobacterial gene coding for phytoene synthase, a carotenoidbiosynthesis enzyme. FEBS Lett 296: 305-310; Ray J A, Bird C R, MaundersM, Grierson D and Schuch W (1987) Sequence of pTOM5, a ripening relatedCDNA from tomato. Nucl Acids Res 15: 10587-10588; Camara B (1993) Plantphytoene synthase complex—component 3 enzymes, immunology, andbiogenesis. Meth Enzymol 214: 352-365]. All the subsequent steps in thepathway occur in membranes. Four desaturation (dehydrogenation)reactions convert phytoene to lycopene via phytofluene, ζ-carotene, andneurosporene. Each desaturation increases the number of conjugateddouble bonds by two such that the number of conjugated double bondsincreases from three in phytoene to eleven in lycopene.

Relatively little is known about the molecular mechanism of theenzymatic dehydrogenation of phytoene [see, Jones B L and Porter J W(1986) Biosynthesis of carotenes in higher plants. CRC Crit Rev PlantSci 3: 295-324; and, Beyer P, Mayer M and Kleinig H (1989) Molecularoxygen and the state of geometric iosomerism of intermediates areessential in the carotene desaturation and cyclization reactions indaffodil chromoplasts. Eur J Biochem 184: 141-150]. It has beenestablished that in cyanobacteria, algae and plants the first twodesaturations, from 15-cis-phytoene to ζ-carotene, are catalyzed by asingle membrane-bound enzyme, phytoene desaturase [see, Jones B L andPorter J W (1986) Biosynthesis of carotenes in higher plants. CRC CritRev Plant Sci 3: 295-324; and, Beyer P, Mayer M and Kleinig H (1989)Molecular oxygen and the state of geometric iosomerism of intermediatesare essential in the carotene desaturation and cyclization reactions indaffodil chromoplasts. Eur J Biochem 184: 141-150]. Since the ζ-caroteneproduct is mostly in the all-trans configuration, a cis-transisomerization is presumed at this desaturation step. The primarystructure of the phytoene desaturase polypeptide in cyanobacteria isconserved (over 65% identical residues) with that of algae and plants[see, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J(1992) A single polypeptide catalyzing the conversion of phytoene toζ-carotene is transcriptionally regulated during tomato fruit ripening.Proc Natl Acad Sci USA 89: 4962-4966; Pecker I, Chamovitz D, Mann V,Sandmann G, Boger P and Hirschberg J (1993) Molecular characterizationof carotenoid biosynthesis in plants: the phytoene desaturase gene intomato. In: Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18.Kluwer, Dordrectht]. Moreover, the same inhibitors block phytoenedesaturase in the two systems [see, Sandmann G and Boger P (1989)Inhibition of carotenoid biosynthesis by herbicides. In: Boger P andSandmann G (eds) Target Sites of Herbicide Action, pp 25-44. CRC Press,Boca Raton, Fla.]. Consequently, it is very likely that the enzymescatalyzing the desaturation of phytoene and phytofluene in cyanobacteriaand plants have similar biochemical and molecular properties, that aredistinct from those of phytoene desaturases in other microorganisms. Onesuch a difference is that phytoene desaturases from Rhodobactercapsulatus, Erwinia sp. or fungi convert phytoene to neurosporene,lycopene, or 3,4-dehydrolycopene, respectively.

Desaturation of phytoene in daffodil chromoplasts [see, Beyer P, Mayer Mand Kleinig H (1989) Molecular oxygen and the state of geometriciosomerism of intermediates are essential in the carotene desaturationand cyclization reactions in daffodil chromoplasts. Eur J Biochem 184:141-150], as well as in a cell free system of Synechococcus sp. strainPCC 7942 [see, Sandmann G and Kowalczyk S (1989) In vitrocarotenogenesis and characterization of the phytoene desaturase reactionin Anacystis. Biochem Biophys Res Com 163: 916-921], is dependent onmolecular oxygen as a possible final electron acceptor, although oxygenis not directly involved in this reaction. A mechanism ofdehydrogenase-electron transferase was supported in cyanobacteria overdehydrogenation mechanism of dehydrogenase-monooxygenase [see, SandmannG and Kowalczyk S (1989) In vitro carotenogenesis and characterizationof the phytoene desaturase reaction in Anacystis. Biochem Biophys ResCom 163: 916-921]. A conserved FAD-binding motif exists in all phytoenedesaturases whose primary structures have been analyzed [see, Pecker I,Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A singlepolypeptide catalyzing the conversion of phytoene to ζ-carotene istranscriptionally regulated during tomato fruit ripening. Proc Natl AcadSci USA 89: 4962-4966; Pecker 1, Chamovitz D, Mann V, Sandmann G, BogerP and Hirschberg J (1993) Molecular characterization of carotenoidbiosynthesis in plants: the phytoene desaturase gene in tomato. In:Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer,Dordrectht]. The phytoene desaturase enzyme in pepper was shown tocontain a protein-bound FAD [see, Hugueney P, Romer S, Kuntz M andCamara B (1992) Characterization and molecular cloning of a flavoproteincatalyzing the synthesis of phytofluene and ζ-carotene in Capsicumchromoplasts. Eur J Biochem 209: 399-407]. Since phytoene desaturase islocated in the membrane, an additional, soluble redox component ispredicted. This hypothetical component could employ NAD(P)⁺, assuggested [see, Mayer M P, Nievelstein V and Beyer P (1992) Purificationand characterization of a NADPH dependent oxidoreductase fromchromoplasts of Narcissus pseudonarcissus—a redox-mediator possiblyinvolved in carotene desaturation. Plant Physiol Biochem 30: 389-398] oranother electron and hydrogen carrier, such as a quinone. The cellularlocation of phytoene desaturase in Synechocystis sp. strain PCC 6714 andAnabaena variabilis strain ATCC 29413 was determined with specificantibodies to be mainly (85%) in the photosynthetic thylakoid membranes[see, Serrano A, Gimenez P, Schmidt A and Sandmann G (1990)Immunocytochemical localization and functional determination of phytoenedesaturase in photoautotrophic prokaryotes. J Gen Microbiol 136:2465-2469].

In cyanobacteria algae and plants ζ-carotene is converted to lycopenevia neurosporene. Very little is known about the enzymatic mechanism,which is predicted to be carried out by a single enzyme [see, Linden H,Vioque A and Sandmann G (1993) Isolation of a carotenoid biosynthesisgene coding for ζ-carotene desaturase from Anabaena PCC 7120 byheterologous complementation. FEMS Microbiol Lett 106: 99-104]. Thededuced amino acid sequence of ζ-carotene desaturase in Anabaena sp.strain PCC 7120 contains a dinucleotide-binding motif that is similar tothe one found in phytoene desaturase.

Two cyclization reactions convert lycopene to β-carotene. Evidence hasbeen obtained that in Synechococcus sp. strain PCC 7942 [see, CunninghamF X Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J (1993) Cloningand functional expression in Escherichia coil of a cyanobacterial genefor lycopene cyclase, the enzyme that catalyzes the biosynthesis ofβ-carotene. FEBS Lett 328: 130-138], as well as in plants [see, Camara Band Dogbo O (1986) Demonstration and solubilization of lycopene cyclasefrom Capsicum chromoplast membranes. Plant Physiol 80: 172-184], thesetwo cyclizations are catalyzed by a single enzyme, lycopene cyclase.This membrane-bound enzyme is inhibited by the triethylamine compounds,CPTA and MPTA [see, Sandmann G and Boger P (1989) Inhibition ofcarotenoid biosynthesis by herbicides. In: Boger P and Sandmann G (eds)Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton,Fla.]. Cyanobacteria carry out only the β-cyclization and therefore donot contain ε-carotene, δ-carotene and α-carotene and their oxygenatedderivatives. The β-ring is formed through the formation of a “carboniumion” intermediate when the C-1,2 double bond at the end of the linearlycopene molecule is folded into the position of the C-5,6 double bond,followed by a loss of a proton from C-6. No cyclic carotene has beenreported in which the 7,8 bond is not a double bond. Therefore, fulldesaturation as in lycopene, or desaturation of at least half-moleculeas in neurosporene, is essential for the reaction. Cyclization oflycopene involves a dehydrogenation reaction that does not requireoxygen. The cofactor for this reaction is unknown. Adinucleotide-binding domain was found in the lycopene cyclasepolypeptide of Synechococcus sp. strain PCC 7942, implicating NAD(P) orFAD as coenzymes with lycopene cyclase.

The addition of various oxygen-containing side groups, such as hydroxy-,methoxy-, oxo-, epoxy-, aldehyde or carboxylic acid moieties, form thevarious xanthophyll species. Little is known about the formation ofxanthophylls. Hydroxylation of β-carotene requires molecular oxygen in amixed-function oxidase reaction.

Clusters of genes encoding the enzymes for the entire pathway have beencloned from the purple photosynthetic bacterium Rhodobacter capsulatus[see, Armstrong G A, Alberti M, Leach F and Hearst J E (1989) Nucleotidesequence, organization, and nature of the protein products of thecarotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol GenGenet 216: 254-268] and from the nonphotosynthetic bacteria Erwiniaherbicola [see, Sandmann G, Woods W S and Tuveson R W (1990)Identification of carotenoids in Erwinia herbicola and in transformedEscherichia coli strain. FEMS Microbiol Lett 71: 77-82; Hundle B S,Beyer P, Kleinig H, Englert H and Hearst J E (1991) Carotenoids ofErwinia herbicola and an Escherichia coli HB101 strain carrying theErwinia herbicola carotenoid gene cluster. Photochem Photobiol 54:89-93; and, Schnurr G, Schmidt A and Sandmann G (1991) Mapping of acarotenogenic gene cluster from Erwinia herbicola and functionalidentification of six genes. FEMS Microbiol Lett 78: 157-162] andErwinia uredovora [see, Misawa N, Nakagawa M, Kobayashi K, Yamano S,Izawa I, Nakamura K and Harashima K (1990) Elucidation of the Erwiniauredovora carotenoid biosynthetic pathway by functional analysis of geneproducts in Escherichia coli. J Bacteriol 172: 6704-6712]. Two genes,al-3 for GGPP synthase [see, Nelson M A, Morelli G, Carattoli A, RomanoN and Macino G (1989) Molecular cloning of a Neurospora crassacarotenoid biosynthetic gene (albino-3) regulated by blue light and theproducts of the white collar genes. Mol Cell Biol 9: 1271-1276; and,Carattoli A, Romano N, Ballario P, Morelli G and Macino G (1991) TheNeurospora crassa carotenoid biosynthetic gene (albino 3). J Biol Chem266: 5854-5859] and al-1 for phytoene desaturase [see, Schmidhauser T J,Lauter F R, Russo V E A and Yanofsky C (1990) Cloning sequencing andphotoregulation of al-1, a carotenoid biosynthetic gene of Neurosporacrassa. Mol Cell Biol 10: 5064-5070] have been cloned from the fungusNeurospora crassa. However, attempts at using these genes asheterologous molecular probes to clone the corresponding genes fromcyanobacteria or plants were unsuccessful due to lack of sufficientsequence similarity.

The first “plant-type” genes for carotenoid synthesis enzyme were clonedfrom cyanobacteria using a molecular-genetics approach. In the firststep towards cloning the gene for phytoene desaturase, a number ofmutants that are resistant to the phytoene-desaturase-specificinhibitor, norflurazon, were isolated in Synechococcus sp. strain PCC7942 [see, Linden H, Sandmann G, Chamovitz D, Hirschberg J and Boger P(1990) Biochemical characterization of Synechococcus mutants selectedagainst the bleaching herbicide norflurazon. Pestic Biochem Physiol 36:46-51]. The gene conferring norflurazon-resistance was then cloned bytransforming the wild-type strain to herbicide resistance [see,Chamovitz D, Pecker I and Hirschberg J (1991) The molecular basis ofresistance to the herbicide norfilurazon. Plant Mol Biol 16: 967-974;Chamovitz D, Pecker I, Sandmann G, Boger P and Hirschberg J (1990)Cloning a gene for norflurazon resistance in cyanobacteria. ZNaturforsch 45c: 482-486]. Several lines of evidence indicated that thecloned gene, formerly called pds and now named crtP, codes for phytoenedesaturase. The most definitive one was the functional expression ofphytoene desaturase activity in transformed Escherichia coli cells [see,Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J and Sandmann G(1991) Functional complementation in Escherichia coli of differentphytoene desaturase genes and analysis of accumulated carotenes. ZNaturforsch 46c: 1045-1051; and, Pecker I, Chamovitz D, Linden H,Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing theconversion of phytoene to ζ-carotene is transcriptionally regulatedduring tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966]. ThecrtP gene was also cloned from Synechocystis sp. strain PCC 6803 bysimilar methods [see, Martinez-Ferez I M and Vioque A (1992) Nucleotidesequence of the phytoene desaturase gene from Synechocystis sp. PCC 6803and characterization of a new mutation which confers resistance to theherbicide norflurazon. Plant Mol Biol 18: 981-983].

The cyanobacterial crtP gene was subsequently used as a molecular probefor cloning the homologous gene from an alga [see, Pecker I, ChamovitzD, Mann V, Sandmann G, Boger P and Hirschberg J (1993) Molecularcharacterization of carotenoid biosynthesis in plants: the phytoenedesaturase gene in tomato. In: Murata N (ed) Research in Photosynthesis,Vol III, pp 11-18. Kluwer, Dordrectht] and higher plants [see, Bartley GE, Viitanen P V, Pecker I, Chamovitz D, Hirschberg J and Scolnik P A(1991) Molecular cloning and expression in photosynthetic bacteria of asoybean cDNA coding for phytoene desaturase, an enzyme of the carotenoidbiosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536; and, PeckerI, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A singlepolypeptide catalyzing the conversion of phytoene to ζ-carotene istranscriptionally regulated during tomato fruit ripening. Proc Natl AcadSci USA 89: 4962-4966]. The phytoene desaturases in Synechococcus sp.strain PCC 7942 and Synechocystis sp. strain PCC 6803 consist of 474 and467 amino acid residues, respectively, whose sequences are highlyconserved (74% identities and 86% similarities). The calculatedmolecular mass is 51 kDa and, although it is slightly hydrophobic(hydropathy index −0.2), it does not include a hydrophobic region whichis long enough to span a lipid bilayer membrane. The primary structureof the cyanobacterial phytoene desaturase is highly conserved with theenzyme from the green alga Dunalliela bardawil (61% identical and 81%similar; [see, Pecker I, Chamovitz D, Mann V, Sandmann G, Boger P andHirschberg J (1993) Molecular characterization of carotenoidbiosynthesis in plants: the phytoene desaturase gene in tomato. In:Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer,Dordrectht]) and from tomato [see, Pecker I, Chamovitz D, Linden H,Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing theconversion of phytoene to ζ-carotene is transcriptionally regulatedduring tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966],pepper [see, Hugueney P, Romer S, Kuntz M and Camara B (1992)Characterization and molecular cloning of a flavoprotein catalyzing thesynthesis of phytofluene and ζ-carotene in Capsicum chromoplasts. Eur JBiochem 209: 399-407] and soybean [see, Bartley G E, Viitanen P V,Pecker I, Chamovitz D, Hirschberg J and Scolnik P A (1991) Molecularcloning and expression in photosynthetic bacteria of a soybean cDNAcoding for phytoene desaturase, an enzyme of the carotenoid biosynthesispathway. Proc Natl Acad Sci USA 88: 6532-6536] (62-65% identical and˜79% similar; [see, Chamovitz D (1993) Molecular analysis of the earlysteps of carotenoid biosynthesis in cyanobacteria: Phytoene synthase andphytoene desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem]).The eukaryotic phytoene desaturase polypeptides are larger (64 kDa);however, they are processed during import into the plastids to matureforms whose sizes are comparable to those of the cyanobacterial enzymes.

There is a high degree of structural similarity in carotenoid enzymes ofRhodobacter capsulatus, Erwinia sp. and Neurospora crassa [reviewed inArmstrong G A, Hundle B S and Hearst J E (1993) Evolutionaryconservation and structural similarities of carotenoid biosynthesis geneproducts from photosynthetic and nonphotosynthetic organisms. MethEnzymol 214: 297-311], including in the crtI gene-product, phytoenedesaturase. As indicated above, a high degree of conservation of theprimary structure of phytoene desaturases also exists among oxygenicphotosynthetic organisms. However, there is little sequence similarity,except for the FAD binding sequences at the amino termini, between the“plant-type” crtP gene products and the “bacterial-type” phytoenedesaturases (crtI gene products; 19-23% identities and 42-47%similarities). It has been hypothesized that crtP and crtl are notderived from the same ancestral gene and that they originatedindependently through convergent evolution [see, Pecker I, Chamovitz D,Linden H, Sandmann G and Hirschberg J (1992) A single polypeptidecatalyzing the conversion of phytoene to ζ-carotene is transcriptionallyregulated during tomato fruit ripening. Proc Natl Acad Sci USA 89:4962-4966]. This hypothesis is supported by the differentdehydrogenation sequences that are catalyzed by the two types of enzymesand by their different sensitivities to inhibitors.

Although not as definite as in the case of phytoene desaturase, asimilar distinction between cyanobacteria and plants on the one hand andother microorganisms is also seen in the structure of phytoene synthase.The crtB gene (formerly psy) encoding phytoene synthase was identifiedin the genome of Synechococcus sp. strain PCC 7942 adjacent to crtP andwithin the same operon [see, Bartley G E, Viitanen P V, Pecker I,Chamovitz D, Hirschberg J and Scolnik P A (1991) Molecular cloning andexpression in photosynthetic bacteria of a soybean cDNA coding forphytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.Proc Natl Acad Sci USA 88: 6532-6536]. This gene encodes a 36-kDapolypeptide of 307 amino acids with a hydrophobic index of −0.4. Thededuced amino acid sequence of the cyanobacterial phytoene synthase ishighly conserved with the tomato phytoene synthase (57% identical and70% similar; Ray J A, Bird C R, Maunders M, Grierson D and Schuch W(1987) Sequence of pTOM5, a ripening related cDNA from tomato. NuclAcids Res 15: 10587-10588]) but is less highly conserved with the crtBsequences from other bacteria (29-32% identical and 48-50% similar withten gaps in the alignment). Both types of enzymes contain two conservedsequence motifs also found in prenyl transferases from diverse organisms[see, Bartley G E, Viitanen P V, Pecker I, Chamovitz D, Hirschberg J andScolnik P A (1991) Molecular cloning and expression in photosyntheticbacteria of a soybean cDNA coding for phytoene desaturase, an enzyme ofthe carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88:6532-6536; Carattoli A, Romano N, Ballario P, Morelli G and Macino G(1991) The Neurospora crassa carotenoid biosynthetic gene (albino 3). JBiol Chem 266: 5854-5859; Armstrong G A, Hundle B S and Hearst J E(1993) Evolutionary conservation and structural similarities ofcarotenoid biosynthesis gene products from photosynthetic andnonphotosynthetic organisms. Meth Enzymol 214: 297-311; Math S K, HearstJ E and Poulter C D (1992) The crtE gene in Erwinia herbicola encodesgeranylgeranyl diphosphate synthase. Proc Natl Acad Sci USA 89:6761-6764; and, Chamovitz D (1993) Molecular analysis of the early stepsof carotenoid biosynthesis in cyanobacteria: Phytoene synthase andphytoene desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem].It is conceivable that these regions in the polypeptide are involved inthe binding and/or removal of the pyrophosphate during the condensationof two GGPP molecules.

The crtQ gene encoding ζ-carotene desaturase (formerly zds) was clonedfrom Anabaena sp. strain PCC 7120 by screening an expression library ofcyanobacterial genomic DNA in cells of Escherichia coli carrying theErwinia sp. crtB and crtE genes and the cyanobacterial crtP gene [see,Linden H, Vioque A and Sandmann G (1993) Isolation of a carotenoidbiosynthesis gene coding for ζ-carotene desaturase from Anabaena PCC7120 by heterologous complementation. FEMS Microbiol Lett 106: 99-104].Since these Escherichia coli cells produce ζ-carotene, brownish-redpigmented colonies that produced lycopene could be identified on theyellowish background of cells producing ζ-carotene. The predictedζ-carotene desaturase from Anabaena sp. strain PCC 7120 is a 56-kDapolypeptide which consists of 499 amino acid residues. Surprisingly, itsprimary structure is not conserved with the “plant-type” (crtP geneproduct) phytoene desaturases, but it has considerable sequencesimilarity to the bacterial-type enzyme (crtI gene product) [see,Sandmann G (1993) Genes and enzymes involved in the desaturationreactions from phytoene to lycopene. (abstract), 10th InternationalSymposium on Carotenoids, Trondheim CL1-2]. It is possible that thecyanobacterial crtQ gene and crtI gene of other microorganismsoriginated in evolution from a common ancestor.

The crtL gene for lycopene cyclase (formerly lcy) was cloned fromSynechococcus sp. strain PCC 7942 utilizing essentially the same cloningstrategy as for crtP. By using an inhibitor of lycopene cyclase,2-(4-methylphenoxy)-triethylamine hydrochloride (MPTA), the gene wasisolated by transformation of the wild-type to herbicide-resistance[see, Cunningham F X Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J(1993) Cloning and functional expression in Escherichia coli of acyanobacterial gene for lycopene cyclase, the enzyme that catalyzes thebiosynthesis of β-carotene. FEBS Lett 328: 130-138]. Lycopene cyclase isthe product of a single gene product and catalyzes the doublecyclization reaction of lycopene to β-carotene. The crtL gene product inSynechococcus sp. strain PCC 7942 is a 46-kDa polypeptide of 411 aminoacid residues. It has no sequence similarity to the crtY gene product(lycopene cyclase) from Erwinia uredovora or Erwinia herbicola.

The gene for β-carotene hydroxylase (crtZ) and zeaxanthin glycosilase(crtX) have been cloned from Erwinia herbicola [see, Hundle B, AlbertiM, Nievelstein V, Beyer P, Kleinig H, Armstrong G A, Burke D H andHearst J E (1994) Functional assignment of Erwinia herbicola Eho10carotenoid genes expressed in Escherichia coli. Mol Gen Genet 254:406-416; Hundle B S, Obrien D A, Alberti M, Beyer P and Hearst J E(1992) Functional expression of zeaxanthin glucosyltransferase fromErwinia herbicola and a proposed diphosphate binding site. Proc NatlAcad Sci USA 89: 9321-9325] and from Erwinia uredovora [see, Misawa N,Nakagawa M, Kobayashi K, Yamano S, Izawa I, Nakamura K and Harashima K(1990) Elucidation of the Erwinia uredovora carotenoid biosyntheticpathway by functional analysis of gene products in Escherichia coli. JBacteriol 172: 6704-6712].

The ketocarotenoid astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione)was first described in aquatic crustaceans as an oxidized form ofβ-carotene. Astaxanthin was later found to be very common in many marineanimals and algae. However, only few animals can synthesize astaxanthinde novo from other carotenoids and most of them obtain it in their food.In the plant kingdom, astaxanthin occurs mainly in some species ofcyanobacteria, algae and lichens. However, it is found rarely also inpetals of higher plant species [see, Goodwin T W (1980) The Biochemistryof the carotenoids, Vol. 1. 2nd Ed, Chapman and Hall, London and NewYork].

The function of astaxanthin as a powerful antioxidant in animals hasbeen demonstrated [see, Miki W (1991) Biological functions andactivities of animal carotenoids. Pure Appl Chem 63: 141]. Astaxanthinis a strong inhibitor of lipid peroxidation and has been shown to playan active role in the protection of biological membranes from oxidativeinjury [see, Palozza P and Krinsky N I (1992) Antioxidant effects ofcarotenoids in vivo and in vitro—an overview. Methods Enzymol 213:403-420; and, Kurashige M, Okimasu E, Inove M and Utsumi K (1990)Inhibition of oxidative injury of biological membranes by astaxanthin.Physiol Chem Phys Med NMR 22: 27]. The chemopreventive effects ofastaxanthin have also been investigated in which astaxanthin was shownto significantly reduce the incidence of induced urinary bladder cancerin mice [see, Tanaka T, Morishita Y, Suzui M, Kojima T, Okumura A. andMori H (1994). Chemoprevention of mouse urinary bladder carcinogenesisby the naturally occurring carotenoid astaxanthin. Carcinogenesis 15:15]. It has also been demonstrated that astaxanthin exertsimmunomodulating effects by enhancing antibody production [see,Jyonouchi H, Zhang L and Tomita Y (1993) Studies of immunomodulatingactions of carotenoids. II. Astaxanthin enhances in vitro antibodyproduction to T-dependent antigens without facilitating polyclonalB-cell activation. Nutr Cancer 19: 269; and, Jyonouchi H, Hill J R,Yoshifumi T and Good R A (1991) Studies of immunomodulating actions ofcarotenoids. I. Effects of β-carotene and astaxanthin on murinelymphocyte functions and cell surface marker expression in-vitro culturesystem. Nutr Cancer 16: 93]. The complete biomedical properties ofastaxanthin remain to be elucidated, but initial results suggest that itcould play an important role in cancer and tumor prevention, as well aseliciting a positive response from the immune system.

Astaxanthin is the principal carotenoid pigment of salmonids and shrimpsand imparts attractive pigmentation in the eggs, flesh and skin [see,Torrisen O J, Hardy R W, Shearer K D (1989) Pigmentation ofsalmonid-carotenoid deposition and metabolism in salmonids. Crit RevAquatic Sci 1: 209]. The world-wide harvest of salmon in 1991 wasapproximately 720,000 MT., of which 25-30% were produced in a variety ofaquaculture facilities [see, Meyers S P (1994) Developments in worldaquaculture, feed formulations, and role of carotenoids. Pure Appl Chem66: 1069]. This is set to increase up to 460,000 MT. by the year 2000[see, Bjorndahl T (1990) The Economics of Salmon Aquaculture. BlackwellScientific, Oxford. pp. 1]. The red coloration of the salmonid fleshcontributes to consumer appeal and therefore affects the price of thefinal product. Animals cannot synthesize carotenoids and they acquirethe pigments through the food chain from the primary producers—marinealgae and phytoplankton. Those grown in intensive culture usually sufferfrom suboptimal color. Consequently, carotenoid-containing nourishmentis artificially added in aquaculture, at considerable cost to theproducer.

Astaxanthin is the most expensive commercially used carotenoid compound(todays-1995 market value is of 2,500-3,500 $/kg). It is utilized mainlyas nutritional supplement which provides pigmentation in a wide varietyof aquatic animals. In the Far-East it is used also for feeding poultryto yield a typical pigmentation of chickens. It is also a desirable andeffective nontoxic coloring for the food industry and is valuable incosmetics. Recently it was reported that astaxanthin is a potentantioxidant in humans and thus is a desirable food additive.

Natural (3S,3′S) astaxanthin is limited in availability. It iscommercially extracted from some crustacea species [see, Torrisen O J,Hardy R W, Shearer K D (1989) Pigmentation of salmonid-carotenoiddeposition and metabolism in salmonids. Crit Rev Aquatic Sci 1: 209].The (3R,3′R) stereoisomer of astaxanthin is produced from Phaffia [ayeast specie, see, Andrewes A G, Phaff H J and Starr M P (1976)Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast.Phytochemistry Vol. 15, pp. 1003-1007]. Synthetic astaxanthin,comprising a 1:2:1 mixture of the (3S,3′S)-, (3S,3′R)- and(3R,3′R)-isomers is now manufactured by Hoffman-La Roche and sold at ahigh price (ca. $2,500/Kg) under the name “CAROPHYLL Pink” [see, Mayer H(1994) Reflections on carotenoid synthesis. Pure & Appl Chem, Vol. 66,pp. 931-938]. Recently a novel gene involved in ketocompoundbiosynthesis, designated crtW was isolated from the marine bacteriaAgrobacterium auranticacum and Alcaligenes PC-1 that produceketocarotenoids such as astaxanthin. When the crtW gene was introducedinto engineered Eschrichia coli that accumulated β-carotene due toErwinia carotenogenic genes, the Escherichia coli transformantssynthesized canthaxanthin a precursor in the synthetic pathway ofastaxanthin [see, Misawa N, Kajiwara S, Kondo K, Yokoyama A, Satomi Y,Saito T, Miki W and Ohtani T (1995) Canthaxanthin biosynthesis by theconversion of methylene to keto groups in a hydrocarbon β-carotene by asingle gene. Biochemical and biophysical research communications Vol.209, pp. 867-876]. It is therefore desirable to find a relativelyinexpensive source of (3S,3′S) astaxanthin to be used as a feedsupplement in aquaculture and as a valuable chemical for various otherindustrial uses.

Although astaxanthin is synthesized in a variety of bacteria, fungi andalgae, the key limitation to the use of biological systems for itsproduction is the low yield of and costly extraction methods in thesesystems compared to chemical synthesis. One way to solve these problemsis to increase the productivity of astaxanthin production in biologicalsystems using recombinant DNA technology. This allows for the productionof astaxanthin in genetically engineered host which, in the case of ahigher plant, is easy to grow and simple to extract. Furthermore,production of astaxanthin in genetically engineered host enables byappropriate host selection to use thus produced astaxanthin in forexample aquaculture applications, devoid of the need for extraction.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a nucleic acid segment which encodesβ-C-4-oxygenase, the enzyme that converts β-carotene to canthaxanthin,as well as recombinant vector molecules comprising a nucleic acidsequence according to the invention, and host cells or transgenicorganisms transformed or transfected with these vector molecules or DNAsegment for the biotechnological production of (3S,3′S) astaxanthin.

Other features and advantages of the invention will be apparent from thefollowing description and from the claims.

SUMMARY OF THE INVENTION

It is a general object of this invention to provide a biotechnologicalmethod for production of (3S,3′S) astaxanthin.

It is a specific object of the invention to provide a peptide having aβ-C-4-oxygenase activity and a DNA segment coding for this peptide toenable a biotechnological production of astaxanthin and otherxanthophylls.

It is a further object of the invention to provide an RNA segmentscoding for a polypeptide comprising an amino acid sequence correspondingto above described peptide.

It is yet a further object of the invention to provide a recombinant DNAmolecule comprising a vector and the DNA segment as described above.

It is still a further object of the invention to provide a host cellcontaining the above described recombinant DNA molecule.

It is another object of the invention to provide a host transgenicorganism containing the above described recombinant DNA molecule or theabove described DNA segment in its cells.

It is still another object of the invention to provide a host transgenicorganism which expresses β-C-4-oxygenase activity in chloroplasts and/orchromoplasts-containing tissues.

It is yet another object of the invention to provide a food additive foranimal or human consumption comprising the above described host cell ortransgenic organism.

It is still another object of the invention to provide a method ofproducing astaxanthin using the above described host cell or transgenicorganism.

It is a further object of the invention to provide a method of producingcanthaxanthin, echinenone, cryptoxanthin, isocryptoxanthinhydroxyechinenone, zeaxanthin, adonirubin, and/or adonixanthin using theabove described host cell or transgenic organism.

Further objects and advantages of the present invention will be clearfrom the description that follows.

In one embodiment, the present invention relates to a DNA segment codingfor a polypeptide comprising an amino acid sequence corresponding toHaematococcus pluvialis crtO gene.

In a further embodiment, the present invention relates to an RNA segmentcoding for a polypeptide comprising an amino acid sequence correspondingto Haematococcus pluvialis crtO gene.

In yet another embodiment, the present invention relates to apolypeptide comprising an amino acid sequence corresponding to aHaematococcus pluvialis crtO gene.

In a further embodiment, the present invention relates to a recombinantDNA molecule comprising a vector and a DNA segment coding for apolypeptide, corresponding to a Haematococcus pluvialis crtO gene.

In another embodiment, the present invention relates to a host cell

In a further embodiment, the present invention relates to a hosttransgenic organism containing the above described recombinant DNAmolecule or the above described DNA segment in its cells.

In another embodiment, the present invention relates to a method of

In yet another embodiment, the present invention relates to a method ofproducing other xanthophylls.

In still another embodiment, the present invention relates to a methodof obtaining high expression of a transgene in plants specifically inchromoplasts-containing cells.

In one further embodiment, the present invention relates to a method ofimporting a carotenoid-biosynthesis enzyme encoded by a transgene intochromoplasts.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawings executed in colorphotograph. Copies of this patent with color photograph(s) will beprovided by the Patent and Trademark Office upon request and payment ofnecessary fee.

The invention herein described, by way of example only, with referenceto the accompanying drawings, wherein:

FIG. 1 is a general biochemical pathway of β-carotene biosynthesis, inwhich pathway all molecules are depicted in an all-trans configuration,wherein IPP is isopentenyl pyrophosphate, DMAPP is dimethylallylpyrophosphate, GPP is geranyl pyrophosphate, FPP is farnesylpyrophosphate, GGPP is geranylgeranyl pyrophosphate and, PPPP isprephytoene pyrophosphate;

FIG. 2 is an identity map between the nucleotide sequence of the crtOcDNA of the present invention (CRTOA.SEQ) and the cDNA cloned byKajiwara et al., (CRTOJ.SEQ) [see, Kajiwara S, Kakizono T, Saito T,Kondo K, Ohtani T, Nishio N, Nagai S and Misawa N (1995) Isolation andfunctional identification of a novel cDNA for astaxanthin biosynthesisfrom Haematococcus pluvialis, and astaxanthin synthesis in Escherichiacoli. Plant Molec Biol 29: 343-352], using a GCG software, wherein (:)indicate identity, (−) indicate a gap and nucleotides numbering isaccording to SEQ ID NO:4 for CRTOA.AMI and Kajiwara et al., forCRTOJ.AMI;

FIG. 3 is an identity map between the amino acid sequence encoded by thecrtO cDNA of the present invention (CRTOA.AMI) and the amino acidsequence encoded by the cDNA cloned by Kajiwara et al., (CRTOJ.AMI)[see, Kajiwara S, Kakizono T, Saito T, Kondo K, Ohtani T, Nishio N,Nagai S and Misawa N (1995) Isolation and functional identification of anovel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli. Plant Molec Biol 29:343-352], using a GCG software, wherein (:) indicate identity, (−)indicate a gap and amino acids numbering is according to SEQ ID NO:4 forCRTOA.AMI and Kajiwara et al., for CRTOJ.AMI;

FIG. 4 is a schematic depiction of a pACYC184 derived plasmid designatedpBCAR and includes the genes crtE, crtB, crtI and crtY of Erwiniaherbicola, which genes are required for production of β-carotene inEscherichia coli cells;

FIG. 5 is a schematic depiction of a pACYC184 derived plasmid designatedpZEAX and includes the genes crtE, crtB, crtI, crtY and crtZ fromErwinia herbicola, which genes are required for production of zeaxanthinin Escherichia coli cells;

FIG. 6 is a schematic depiction of a pBluescriptSK⁻ derived plasmiddesignated pHPK, containing a full length cDNA insert encoding aβ-carotene C-4-oxygenase enzyme from Haematococcus pluvialis, designatedcrtO and set forth in SEQ ID NO:1, which cDNA was identified by colorcomplementation of Escherichia coli cells;

FIG. 7 is a schematic depiction of a pACYC 184 derived plasmiddesignated pCANTHA which was derived by inserting a 1.2 kb PstI—PstI DNAfragment, containing the cDNA encoding the β-C-4-oxygenase fromHaematococcus pluvialis isolated from the plasmid pHPK of FIG. 6 andinserted into a PstI site in the coding sequence of the crtZ gene in theplasmid pZEAX of FIG. 5; this recombinant plasmid carries the genescrtE, crtB, crtI, crtY of Erwinia herbicola and the crtO gene ofHaematococcus pluvialis, all required for production of canthaxanthin inEscherichia coil cells;

FIG. 8 is a schematic depiction of a pACYC184 derived plasmid designatedpASTA which was derived by inserting the 1.2 kb PstI—PstI DNA fragment,containing the cDNA of the β-C-4-oxygenase from Haematococcus pluvialisisolated from the plasmid pHPK of FIG. 6 and inserted into a PstI sitewhich exists 600 bp downstream of the crtE gene in the plasmid pZEAX ofFIG. 5; this recombinant plasmid carries the genes crtE, crtB, crtI,crtY, crtZ of Erwinia herbicola and the crtO gene of Haematococcuspluvialis, all required for production of astaxanthin in Escherichiacoli cells;

FIG. 9 is a schematic depiction of a pBR328 derived plasmid designatedPAN3.5-KETO which was derived by inserting the 1.2 kb PstI—PstI DNAfragment, containing the cDNA of the β-C-4-oxygenase from Haematococcuspluvialis isolated from the plasmid pHPK of FIG. 6 and inserted into aPstI site which exists in a β-lactamase gene in a plasmid designatedpPAN35D5 [described in Hirschberg J, Ohad N, Pecker I and Rahat A (1987)Isolation and characterization of herbicide resistant mutants in thecyanobacterium Synechococcus R2. Z. Naturforsch 42c: 102-112], whichcarries the psbAI gene from the cyanobacterium Synechococcus PCC7942 inthe plasmid vector pBR328 [see, Hirschberg J, Ohad N, Pecker I and RahatA (1987) Isolation and characterization of herbicide resistant mutantsin the cyanobacterium Synechococcus R2. Z. Naturforsch 42c: 102-112];this recombinant plasmid carries the crtO gene of Haematococcuspluvialis, required for production of astaxanthin in SynechococcusPCC7942 cells;

FIG. 10 is a schematic depiction of the T-DNA region of a Ti binaryplasmid (E. coli, Agrobacterium) designated pBIB [described by Becker D(1990) Binary vectors which allow the exchange of plant selectablemarkers and reporter genes. Nucleic Acids Research 18:230] which is aderivative of the Ti plasmid pBI101 [described by Jeffesrson A R,Kavanagh T A and Bevan W M (1987) GUS fusions: β-glucuronidase as asensitive and versatile gene fusion marker in higher plants. The EMBO J.6: 3901-3907], wherein B_(R) and B_(L) are the right and left borders,respectively, of the T-DNA region, pAg7 is the polyadenylation site ofgene 7 of Agrobacterium Ti-plasmid, pAnos is a 250 bp long DNA fragmentcontaining the poly adenylation site of the nopaline synthase gene ofAgrobacterium, NPT II is a 1,800 bp long DNA fragment coding forkanamycin resistance, pnos is a 300 bp long DNA fragment containing thepromoter sequence of the nopaline synthase gene of Agrobacterium,whereas pAnos is a 300 bp long DNA fragment containing the polyadenylation site of the nopaline synthase gene of Agrobacterium;

FIG. 11 is a schematic depiction of the T-DNA region of a Ti binaryplasmid (E. coli, Agrobacterium) designated pPTBIB which was prepared bycloning a genomic DNA sequence of a tomato species Lycopersiconesculentum marked PT (nucleotides 1 to 1448 of the Pds gene as publishedin Mann V, Pecker I and Hirschberg J (1994) cloning and characterizationof the gene for phytoene desaturase (Pds) from tomato (Lycopersiconesculentum). Plant Molecular Biology 24: 429-434), which contains thepromoter of the Pds gene and the coding sequence for the amino terminusregion of the polypeptide PDS that serve as a transit peptide for importinto chloroplasts and chromoplasts, into a HindIII-SmaI site of thebinary plasmid vector pBIB of FIG. 10, wherein B_(R) and B_(L), pAg7,pAnos, NPT II, pnos and pAnos are as defined above;

FIG. 12 is a schematic depiction of the T-DNA region of a Ti binaryplasmid (E. coli, Agrobacterium) designated pPTCRTOBIB which wasprepared by cloning a 1,110 nucleotide long Eco47III-NcoI fragment ofthe cDNA of crtO from H. pluvialis (nucleotides 211 to 1321 of SEQ IDNO:1) into the SmaI site of the plasmid pPTBIB of FIG. 11, such that thecoding nucleotide sequence of the amino terminus of PDS is in the samereading frame of crtO, wherein B_(R) and B_(L), pAg7, pAnos, NPT II,pnos, and pAnos are as defined above, PT is the promoter and transitpeptide coding sequences of Pds from tomato and CRTO is the nucleotidesequence of crtO from H. pluvialis (nucleotides 211 to 1321 of SEQ IDNO:1);

FIG. 13 shows a Southern DNA blot analysis of HindIII-digested genomicDNA extracted from wild type (WT) and crtO tobacco transgenic plants,designated 2, 3, 4, 6, 9 and 10, according to the present invention,using the crtO cDNA as a radioactive probe essentially as described inSambrook et al., Molecular Cloning; A Laboratory Manual. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. 1989, wherein the size ofmarker (M) DNA fragments in kilobase pairs (kb) is indicated on the leftas well as the expected position (arrow) of an internal T-DNA HindIIIfragment as was deduced from the sequence of pPTPDSBIB shown in FIG. 12which contain the crtO cDNA sequence;

FIG. 14 shows a biosynthesis pathway of astaxanthin;

FIG. 15 shows a flower from a wild type tobacco plant and a flower froma transgenic tobacco plant according to the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is, in general, of a biotechnological method forproduction of (3S,3′S) astaxanthin. In particular, the present inventionis of a peptide having a β-C-4-oxygenase activity; a DNA segment codingfor this peptide; an RNA segments coding for this peptide; a recombinantDNA molecule comprising a vector and the DNA segment; a host cell ororganism containing the above described recombinant DNA molecule or DNAsegment; and of a method for biotechnologically producing (3S,3′S)astaxanthin or a food additive containing (3S,3′S) astaxanthin, usingthe host.

The unicellular fresh-water green alga Haematococcus pluvialisaccumulates large amounts of (3S,3′S) astaxanthin when exposed tounfavorable growth conditions, or following different environmentalstresses such as phosphate or nitrogen starvation, high concentration ofsalt in the growth medium or high light intensity [see, Yong Y Y R andLee Y K (1991) Phycologia 30 257-261; Droop M R (1954) Arch Microbiol20: 391-397; and, Andrewes A. G, Borch G, Liaaen-Jensen S and SnatzkeG.(1974) Acta Chem Scand B28: 730-736]. During this process, thevegetative cells of the alga form cysts and change their color fromgreen to red. The present invention discloses the cloning of a cDNA fromHaematococcus pluvialis, designated crtO, which encodes aβ-C-4-oxygenase, the enzyme that converts β-carotene to canthaxanthin,and its expression in a heterologous systems expressing β-carotenehydroxylase (e.g., Erwinia herbicola crtZ gene product), leading to theproduction of (3S,3′S) astaxanthin.

The crtO cDNA and its encoded peptide having a β-C-4-oxygenase activityare novel nucleic and amino acid sequences, respectively. The cloningmethod of the crtO cDNA took advantage of a strain of Escherichia coli,which was genetically engineered to produce β-carotene, to which a cDNAlibrary of Haematococcus pluvialis was transfected and expressed. Visualscreening for brown-red pigmented Escherichia coli cells has identifieda canthaxanthin producing transformant. Thus cloned cDNA has beenexpressed in two heterologous systems (Escherichia coli andSynechococcus PCC7942 cells) both able to produce β-carotene and furtherinclude an engineered (Erwinia herbicola crtZ gene product) orendogenous β-carotene hydroxylase activity, and was shown to enable theproduction of (3S,3′S) astaxanthin in both these systems.

The crtO cDNA or its protein product exhibit no meaningful nucleic- oramino acid sequence similarities to the nucleic- or amino acid sequenceof crtW and its protein product isolated from the marine bacteriaAgrobacterium auranticacum and Alcaligenes PC-1 that produceketocarotenoids such as astaxanthin [see, Misawa N, Kajiwara S, Kondo K,Yokoyama A, Satomi Y, Saito T, Miki W and Ohtani T (1995) Canthaxanthinbiosynthesis by the conversion of methylene to keto groups in ahydrocarbon β-carotene by a single gene. Biochemical and biophysicalresearch communications Vol. 209, pp. 867-876].

However, the crtO cDNA and its protein product exhibit substantialnucleic- and amino acid sequence identities with the nucleic- and aminoacid sequence of a recently cloned cDNA encoding a 320 amino acidsprotein product having β-carotene oxygenase activity, isolated fromHaematococcus pluvialis [see, Kajiwara S, Kakizono T, Saito T, Kondo K,Ohtani T, Nishio N, Nagai S and Misawa N (1995) Isolation and functionalidentification of a novel cDNA for astaxanthin biosynthesis fromHaematococcus pluvialis, and astaxanthin synthesis in Escherichia coli.Plant Molec Biol 29: 343-352]. Nevertheless, as presented in FIG. 2 thedegree of sequence identity between the crtO cDNA (CRTOA.SEQ in FIG. 2)and the cDNA described by Kajiwara et al. (CRTOJ.SEQ in FIG. 2) [seereference above] is 75.7% and, as presented in FIG. 3 the degree ofsequence identity between the crtO cDNA protein product (CRTOA.AMI inFIG. 3) and the protein described by Kajiwara et al. (CRTOJ.AMI in FIG.3) is 78%, as was determined using a GCG software.

As will be described in details hereinbelow, the crtO cDNA can thus beemployed to biotechnologically produce (3S,3′S) astaxanthin in systemswhich are either easy to grow and can be used directly as an additive tofish food, or systems permitting a simple and low cost extractionprocedure of astaxanthin.

In one embodiment, the present invention relates to a DNA segment codingfor a polypeptide comprising an amino acid sequence corresponding toHaematococcus pluvialis crtO gene and allelic and species variations andfunctional naturally occurring and/or man-induced variants thereof. Thephrase 'allelic and species variations and functional naturallyoccurring and/or man-induced variants' as used herein and in the claimsbelow refer to the source of the DNA (or RNA as described below) ormeans known in the art for obtaining it. However the terms ‘variation’and ‘variants’ indicate the presence of sequence dissimilarities (i.e.,variations). It is the intention herein and in the claims below that thesequence variations will be 77-80%, preferably 80-85%, more preferably85-90%, most preferably 90-100% of identical nucleotides. In a preferredembodiment the DNA segment comprises the sequence set forth in SEQ IDNO:1. In another preferred embodiment, the DNA segment encodes the aminoacid sequence set forth in SEQ ID NO:4.

The invention also includes a pure DNA segment characterized asincluding a sequence which hybridizes under high stringency conditions[e.g., as described in Sambrook et al., Molecular Cloning; A LaboratoryManual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989] toa nucleic acid probe which includes at least fifteen, preferably atleast fifty, more preferably at least hundred, even more preferably atleast two hundred, even more preferably at least five hundred successivenucleotides of SEQ ID NO:1 or SEQ ID NO:2. Alternatively, the DNAsegment of the invention may be characterized as being capable ofhybridizing under low-stringent conditions to a nucleic acid probe whichincludes the coding sequence (nucleotides 166 through 1152) of SEQ IDNO:1 or SEQ ID NO:2. An example of such low-stringency conditions is asdescribed in Sambrook et al., using a lower hybridization temperature,such as, for example, 20° C. below the temperature employed forhigh-stringency hybridization conditions, as described above.

The DNA segment of the invention may also be characterized as beingcapable of hybridizing under high-stringent conditions to a nucleic acidprobe which includes the coding sequence (nucleotides 166 through 1152)of SEQ ID NO:1 or SEQ ID NO:2.

The invention also includes a synthetically produced oligonucleotide(e.g., oligodeoxyribonucleotide or oligoribonucleotide and analogsthereof) capable of hybridizing with at least ten-nucleotide segments ofSEQ ID NO:1 or SEQ ID NO:2.

In another embodiment, the present invention relates to an RNA segmentcoding for a polypeptide comprising an amino acid sequence correspondingto Haematococcus pluvialis crtO gene and allelic and species variationsand functional naturally occurring and/or man-induced variants thereof.In a preferred embodiment the RNA segment comprises the sequence setforth in SEQ ID NO:2. In another preferred embodiment, the RNA segmentencodes the amino acid sequence set forth in SEQ ID NO:4.

The invention also includes a pure RNA characterized as including asequence which hybridizes under high stringent conditions to a nucleicacid probe which includes at least at least fifteen, preferably at leastfifty, more preferably at least hundred, even more preferably at leasttwo hundred, even more preferably at least five hundred succsesivenucleotides of SEQ ID NO:1 or SEQ ID NO:2. Alternatively, the RNA of theinvention may be characterized as being capable of hybridizing underlow-stringent conditions to a nucleic acid probe which includes thecoding sequence (nucleotides 166 through 1152) of SEQ ID NO:1 or SEQ IDNO:2. Additionally, the RNA of the invention may be characterized asbeing capable of hybridizing under high-stringent conditions to anucleic acid probe which includes the coding sequence (nucleotides 166through 1152) of SEQ ID NO:1 or SEQ ID NO:2.

In another embodiment, the present invention relates to a polypeptidecomprising an amino acid sequence corresponding to a Haematococcuspluvialis crtO gene and allelic, species variations and functionalnaturally occurring and/or man-induced variants thereof. In a preferredembodiment, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO:4.

It should be noted that the invention includes any peptide which ishomologous (i.e., 80-85%, preferably 85-90%, more preferably 90-100% ofidentical amino acids) to the above described polypeptide. The term‘homologous’ as used herein and in the claims below, refers to thesequence identity between two peptides. When a position in both of thetwo compared sequences is occupied by identical amino acid monomericsubunits, it is homologous at that position. The homology between twosequences is a function of the number of homologous positions shared bythe two sequences. For example, if eight of ten of the positions in twosequences are occupied by identical amino acids then the two sequencesare 80% homologous.

Other polypeptides which are also included in the present invention areallelic variations, other species homologs, natural mutants, inducedmutants and peptides encoded by DNA that hybridizes under high or lowstringency conditions (see above) to the coding region (nucleotides 166through 1152) of SEQ ID NO:1 or SEQ ID NO:2.

In another embodiment, the present invention relates to a recombinantDNA molecule comprising a vector (for example plasmid or viral vector)and a DNA segment coding for a polypeptide, as described above. In apreferred embodiment, the DNA segment is present in the vector operablylinked to a promoter.

In a further embodiment, the present invention relates to a host cellcontaining the above described recombinant DNA molecule or DNA segment.Suitable host cells include prokaryotes (such as bacteria, includingEscherichia coli) and both lower eukaryotes (for example yeast) andhigher eukaryotes (for example, algae, plant or animal cells).Introduction of the recombinant molecule into the cell can be effectedusing methods known in the art such as, but not limited to,transfection, transformation, micro-injection, gene bombardment etc. Thecell thus made to contain the above described recombinant DNA moleculesmay be grown to form colonies or may be made to differentiate to form adifferentiated organism. The recombinant DNA molecule may be transientlycontained (e.g., by a process known in the art as transienttransfection) in the cell, nevertheless, it is preferred that therecombinant DNA molecule is stably contained (e.g., by a process knownin the art as stable transfection) in the cell. Yet in a preferredembodiment the cell is endogenously producing, or is made by geneticengineering means to produce, β-carotene, and the cell containsendogenous or genetically engineered β-carotene hydroxylase activity.Such a cell may be used as a food additive for animal (e.g., salmon) andhuman consumption. Furthermore, such a cell may be used for extractingastaxanthin and/or other xanthophylls, as described hereinbelow.

In a further embodiment, the present invention relates to a hosttransgenic organism (e.g., a higher plant or animal) containing theabove described recombinant DNA molecule or the above described DNAsegment in its cells. Introduction of the recombinant molecule or theDNA segment into the host transgenic organism can be effected usingmethods known in the art. Yet, in a preferred embodiment the hostorganism is endogenously producing, or is made by genetic engineeringmeans to produce, β-carotene and, also preferably the host organismcontains endogenous or genetically engineered β-carotene hydroxylaseactivity. Such an organism may be used as a food additive for animal(e.g., salmon) and human consumption. Furthermore, such an organism maybe used for extracting astaxanthin and/or other xanthophylls, asdescribed hereinbelow.

In another embodiment, the present invention relates to a method ofproducing astaxanthin using the above described host cell or transgenicorganism. In yet another embodiment, the present invention relates to amethod of producing xanthophylls such as canthaxanthin, echinenone,cryptoxanthin, isocryptoxanthin, hydroxyechinenone, zeaxanthin,adonirubin, 3-hydroxyechinenone, 3′-hydroxyechinenone and/oradonixanthin using the above described host cell or transgenic organism.For these purposes provided is a cell or a transgenic organism asdescribed above. The host cell or organism are made to grow underconditions favorable of producing astaxanthin and the above listedadditional xanthophylls which are than extracted by methods known in theart.

In yet another embodiment, the present invention relates to a transgenicplant expressing a transgene coding for a polypeptide including an aminoacid sequence corresponding to Haematococcus pluvialis crtO gene,allelic and species variants or functional naturally occurring orman-induced variants thereof. Preferably the expression is highest inchromoplasts-containing tissues.

In yet another embodiment, the present invention relates to arecombinant DNA vector which includes a first DNA segment encoding apolypeptide for directing a protein into plant chloroplasts orchromoplasts (e.g., derived from the Pds gene of tomato) and an in framesecond DNA segment encoding a polypeptide including an amino acidsequence corresponding to Haematococcus pluvialis crtO gene, allelic andspecies variants or functional naturally occurring and man-inducedvariants thereof.

In yet another embodiment, the present invention relates to arecombinant DNA vector which includes a first DNA segment including apromoter highly expressible in plant chloroplasts orchromoplasts-containing tissues (e.g., derived from the Pds gene oftomato) and a second DNA segment encoding a polypeptide including anamino acid sequence corresponding to Haematococcus pluvialis crtO gene,allelic and species variants or functional naturally occurring andman-induced variants thereof.

Reference in now made to the following examples, which together with theabove descriptions, illustrate the invention.

EXAMPLES

The following protocols and experimental details are referenced in theExamples that follow:

Algae and growth conditions. Haematococcus pluvialis (strain 34/7 fromthe Culture Collection of Algae and Protozoa, Windermere, UK) was kindlyprovided by Dr. Andrew Young from the Liverpool John Moores University.Suspension cultures of the alga were grown in a liquid medium asdescribed by Nichols and Bold [see, Nichols H W, Bold H C (1964)Trichsarcina polymorpha gen et sp nov J Phycol 1: 34-39]. For inductionof astaxanthin biosynthesis cells were harvested, washed in water andresuspended in a nitrogen-depleted medium. The cultures were maintainedin 250 ml Erlenmeyer flasks under continuous light (photon flux of 75μE/m²/s), at 25° C., on a rotary shaker at 80 rpm.

Construction of cDNA library. The construction of a cDNA library fromHaematococcus pluvialis was described in detail by Lotan and Hirschberg(1995) FEBS letters 364: 125-128. Briefly, total RNA was extracted fromalgal cells grown for 5 days under nitrogen-depleted conditions (cellcolor brown-red). Cells from a 50 ml culture were harvested and theirRNA content was extracted using Tri reagent (Molecular Research Center,INC.). Poly-An RNA was isolated by two cycles of fractionation on oligodT-cellulose (Boehringer). The final yield was 1.5% of the total RNA.The cDNA library was constructed in a Uni-ZAP™ XR vector, using aZAP-cDNA synthesis kit (both from Stratagene). Escherichia coli cells ofstrain XL1-Blue MRF′ (Stratagene) were used for amplification of thecDNA library.

Plasmids and Escherichia coli strains. Plasmid pPL376, which containsthe genes necessary for carotenoid biosynthesis in the bacterium Erwiniaherbicola was obtained from Tuveson [for further details regardingplasmid pPL376 see, Tuveson R W, Larson R A & Kagan J (1988) Role ofcloned carotenoid genes expressed in Escherichia coli in protectingagainst inactivation by near-UV light and specific phototoxic molecules.J Bacteriol 170: 4675-4680]. Cells of Escherichia coli strain JM109 thatcarry the plasmid pPL376 accumulate the bright yellow carotenoid,zeaxanthin glycoside. In a first step, a 1.1 kb SalI—SalI fragment wasdeleted from this plasmid to inactivate the gene crtX, coding forzeaxanthin glucosyl transferase. In a second step, partial BamHIcleavage of the plasmid DNA, followed by self ligation, deleted a 0.8 kbfragment which inactivated crtZ, encoding β-carotene hydroxylase. Apartial BglII cleavage generated a fragment of 7.4 kb which was clonedin the BamHI site of the plasmid vector pACYC 184. As shown in FIG. 4,the resulting recombinant plasmid, which carried the genes crtE, crtB,crtI and crtY, was designated pBCAR [Lotan and Hirschberg (1995) FEBSletters 364: 125-128].

Plasmid pBCAR was transfected into SOLR strain cells of Escherichia coli(Stratagene). Colonies that appeared on chloramphenicol-containing LuriaBroth (LB) medium [described in Sambrook et al., Molecular Cloning; ALaboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. 1989], carried this plasmid and developed a deep yellow-orangecolor due to the accumulation of β-carotene.

As shown in FIG. 5, an additional plasmid, designated pZEAX, whichallows for zeaxanthin synthesis and accumulation in Escherichia coli wasconstructed [this plasmid is described in details in Lotan andHirschberg (1995) FEBS letters 364: 125-128]. SOLR strain Escherichiacoli cells were used as a host for the pZEAX plasmid. Escherichia colicells were grown on LB medium (see above), at 37° C. in the dark on arotary shaker at 225 rpm. Ampicillin (50 μg/ml) and/or chloramphenicol(30μ/ml) (both from Sigma) were added to the medium for selection ofappropriate transformed cells.

As shown in FIG. 6, a plasmid, pHPK, containing the full length cDNA ofthe β-carotene C-4-oxygenase enzyme was identified by colorcomplementation as described by Lotan and Hirschberg (1995) FEBS letters364: 125-128 (see description herein below). A 1.2 kb PstI—PstI DNAfragment, containing the cDNA of the β-C-4-oxygenase from Haematococcuspluvialis, was isolated from plasmid pHPK and inserted into a PstI sitein the coding sequence of the crtZ gene in the plasmid pZEAX. Thisrecombinant plasmid was designated pCANTHA and is shown in FIG. 7.

The same 1.2 kb PstI—PstI fragment was also inserted into a PstI sitewhich exists 600 bp downstream of the crtE gene in the plasmid pZEAX.The resulting recombinant plasmid was designated pASTA and is shown inFIG. 8.

The same 1.2 kb PstI—PstI fragment was also inserted into a PstI sitewhich exists in the β-lactamase gene in the plasmid pPAN35D5 [HirschbergJ, Ohad N, Pecker I and Rahat A (1987) Isolation and characterization ofherbicide resistant mutants in the cyanobacterium Synechococcus R2. Z.Naturforsch 42c: 102-112], which carries the psbAI gene from thecyanobacterium Synechococcus PCC7942 in the plasmid vector pBR328[Hirschberg J, Ohad N, Pecker I and Rahat A (1987) Isolation andcharacterization of herbicide resistant mutants in the cyanobacteriumSynechococcus R2. Z. Naturforsch 42c: 102-112]. This plasmid wasdesignated PAN3.5-KETO and is shown in FIG. 9. This plasmid was used inthe transformation of Synechococcus PCC7942 cells following proceduresdescribed by Golden [Golden SS (1988) Mutagenesis of cyanobacteria byclassical and gene-transfer-based methods. Methods Enzymol 167:714-727].

Excision of phage library and screening for a β-carotene oxygenase gene.Mass excision of the cDNA library, which was prepared as describedhereinabove, was carried out using the ExAssist helper phage(Stratagene) in cells of SOLR strain of Escherichia coli that carriedthe plasmid pBCAR. The excised library in phagemids form was transfectedinto Escherichia coli cells strain XL1-Blue and the cells were plated onLB plates containing 1 mM isopropylthio-β-D-galactosidase (IPTG), 50μg/ml ampicillin and 30 μg/ml chloramphenicol, in a density that yieldedapproximately 100-150 colonies per plate. The plates were incubated at37° C. overnight and further incubated for two more days at roomtemperature. The plates were then kept at 4° C. until screened forchanges in colony colors.

A plasmid for high expression of crtO in chromoplasts. As shown in FIGS.10-11, a genomic DNA sequence of a tomato species Lycopersiconesculentum (nucleotides 1 to 1448 of the Pds gene [as published in MannV, Pecker I and Hirschberg J (1994) cloning and characterization of thegene for phytoene desaturase (Pds) from tomato (Lycopersiconesculentum). Plant Molecular Biology 24: 429-434], which contains thepromoter of the Pds gene and the coding sequence for the amino terminusregion of the polypeptide PDS that serve as a transit peptide for importinto chloroplasts and chromoplasts, was cloned into a HindIII-SmaI siteof the binary plasmid vector pBIB, [described by Becker D (1990) Binaryvectors which allow the exchange of plant selectable markers andreporter genes. Nucleic Acids Research 18: 230], shown in FIG. 10. Therecombinant plasmid was designated pPTBIB and is shown in FIG. 11.

As shown in FIG. 12, a 1,110 nucleotide long Eco47III-NcoI fragment,containing the cDNA of crtO from H. pluvialis (nucleotides 211 to 1321of SEQ ID NO:1) was sub-cloned into the SmaI site of the plasmid pPTBIB(FIG. 11) so that the coding nucleotide sequence of the amino terminusof Pds is in the same reading frame as crtO. The recombinant plasmid wasdesignate pPTCRTOBIB.

Formation of transgenic higher plant. The DNA of pPTCRTOBIB wasextracted from E. coli cells and was transferred into cells ofAgrobacterium tumefaciens strain EHA105 [described by Hood E E, Gelvin SB, Melchers L S and Hoekema A (1993) Transgenic Research 2: 208-218]using electroporation as described for E. coli [Dower J W, Miller F Jand Ragdsale W C (1988) High efficiency transformation of E. coli byhigh voltage electroporation. Nuc. Acids Res. 18: 6127-6145].Agrobacterium cells were grown at 28° C. in LB medium supplemented with50 μg/ml streptomycin and 50 μg/ml kanamycin as selective agents. Cellsof Agrobacterium carrying pPTCRTOBIB were harvested from a suspensionculture at the stationary phase of growth and used for transformation asdescribed by Horsch R B, Fry J E, Hoffmann N L, Eicholtz D, Rogers S Gand Fraley R T, A simple and general method for transferring genes intoplants. Science (1985) 227: 1229-1231; and Jeffesrson A R, Kavanagh T Aand Bevan W M (1987) GUS fusions: β-galucuronidase as a sensitive andversatile gene fusion marker in higher plants. The EMBO J. 6: 3901-3907.

Leaf explants of Nicotiana taabaccum strain NN were infected with thetransformed Agrobacterium cells and kanamycin-resistant transgenicplants were regenerated according to protocols described by Horsch etal. (1985) and Jefferson et al. (1987) cited above.

With reference now to FIG. 13, the presence of the DNA sequence of thecrtO gene-construct in the fully developed regenerated plants wasdetermined by DNA Southern blot analysis. To this end DNA was extractedfrom the leaves [according to a protocol described by Kanazawa andTsutsumi (1992) Extraction of restrictable DNA from plants of the genusNelumbo. Plant Molecular Biology Reports 10: 316-318], digested with theendonuclease HindIII, the fragments were size separated by gelelectrophoresis and hybridized with radioactively labeled crtO sequence(SEQ ID NO: 1).

It was determined that each transgenic plant that was examined containedat least one copy of the crtO DNA sequence, yielding a 1.75 kb band(arrow), originating from an internal HindIII-HindIII fragment of theT-DNA of pPTCRTOBIB, additional bands originating from partialdigestion, additional band/s whose sizes vary, depending on the positionof insertion in the plant genome and a 1.0 kb band originating from thetobacco plant itself which therefore also appears in the negativecontrol WT lane.

Sequence analysis. DNA sequence analysis was carried out by the dideoxymethod [see, Sanger F, Nicklen S & Coulsen A R (1977) DNA sequencingwith chain termination inhibitors. Proc Natl Acad Sci USA 74:5463-5467].

Carotenoids analysis. Aliquots of Escherichia coli cells which weregrown in liquid in LB medium were centrifuged at 13,000 g for 10minutes, washed once in water and re-centrifuged. After removing thewater the cells were resuspended in 70 μl of acetone and incubated at65° C. for 15 minutes. The samples were centrifuged again at 13,000 gfor 10 minutes and the carotenoid-containing supernatant was placed in aclean tube. The carotenoid extract was blown to dryness under a streamof nitrogen (N₂) gas and stored at −20° C. until required for analysis.Carotenoids from plant tissues were extracted by mixing 0.5-1.0 gr oftissue with 100 μl of acetone followed by incubation at 65° C. for 15minutes and then treating the samples as described above.

High-performance liquid chromatography (HPLC) of the carotenoid extractswas carried out using an acidified reverse-phase C₁₈ column, SpherisorbODS-2 (silica 5 μm 4.6 mm×250 mm) (Phenomenex®). The mobile phase waspumped by triphasic Merck-Hitachi L-6200A high pressure pumps at a flowrate of 1.5 ml/min. The mobile phase consisted of an isocratic solventsystem comprised of hexane/dichloromethane/isopropylalcohol/triethylamine (88.5:10:1.5:0.1, v/v). Peaks were detected at 470nm using a Waters 996 photodiode-array detector. Individual carotenoidswere identified by their retention times and their typical absorptionspectra, as compared to standard samples of chemically pure β-carotene,zeaxanthin, echinenone, canthaxanthin, adonirubin and astaxanthin (Thelatter four were kindly provided by Dr. Andrew Young from Liverpool JohnMoores University).

Thin layer chromatography (TLC) was carried out using silica gel 60 F₂₅₄plates (Merck), using ethyl acetate/benzene (7:3, v/v) as an eluent.Visible absorption spectra were recorded with a Shimadzu UV-160 Aspectrophotometer. All spectra were recorded in acetone. Spectral finestructure was expressed in terms of %III/II [Britton, G. (1995).UV/Visible Spectroscopy. In: Carotenoids; Vol IB, Spectroscopy. Eds.Britton G, Liaaen-Jensen S and Pfander H. Birkhauser Verlag, Basel. pp.13-62].

Isolation and identification of the carotenoids extracted from cells ofE. coli are treated in order of increasing adsorption (decreasing R_(f)values) on silica TLC plates. Carotenoids structure and the biosynthesispathway of astaxanthin are given in FIG. 14. The following details referto the carotenoids numbered 1 through 9 in FIG. 14.

β-Carotene (1). R_(f)0.92 inseparable from authentic (1). R_(t).VISλ_(max) nm: (428), 452, 457, % III/II=0.

Echinenone (2). R_(f)0.90 inseparable from authentic (2). R_(t).VISλ_(max) nm: 455, %III/II=0.

Canthaxanthin (3). R_(f)0.87.inseparable from authentic (3). R_(t).VISλ_(max) nm: 470, %III/II=0.

β-Cryptoxanthin (4). R_(f)0.83. R_(t).VIS λ_(max) nm: (428), 451, 479,%III/II=0.

Adonirubin (5). R_(f)0.82 inseparable from authentic (5). R_(t).VISλ_(max) nm: 476, %III/II=0.

Astaxanthin (6). R_(f)0.79 inseparable from authentic (6). R_(t).VISλ_(max) nm: 477, %III/II=0.

Adonixanthin (7). R_(f)0.72. R_(t).VIS λ_(max) nm: 464, %III/II=0.

Zeaxanthin (8). R_(f)0.65 inseparable from authentic (8). R_(t).VISλ_(max) nm: (428), 451, 483, %III/II=27.

Hydroxyechinenone (9). R_(f)80, R_(t), 3.0.VIS λ_(max) nm: 464,%III/II=0.

Chirality configuration. Chirality configuration of astaxanthin wasdetermined by HPLC of the derived diastereoisomeric camphanates of theastaxanthin [Renstrom B, Borch G, Skulberg M and Liaaen-Jensen S (1981)Optical purity of (3S,3S)-astaxanthin from Haematococcus pluvialis.Phytochem 20: 2561-2565]. The analysis proved that the Escherichia colicells synthesize pure (3S,3′S) astaxanthin.

Example 1 Cloning the βC-4-oxygenase Gene

A cDNA library was constructed in Lambda ZAP II vector from poly-An RNAof Haematococcus pluvialis cells that had been induced to synthesizeastaxanthin by nitrogen deprivation as described hereinabove. The entirelibrary was excised into β-carotene-accumulating cells of Escherichiacoli, strain SOLR, which carried plasmid pBCAR (shown in FIG. 4).Screening for a β-carotene oxygenase gene was based on colorvisualization of colonies of size of 3 mm in diameter. Astaxanthin andother oxygenated forms of β-carotene (i.e., xanthophylls) have distinctdarker colors and thus can be detected from the yellow β-carotenebackground. The screening included approximately 100,000 colonies whichwere grown on LB medium plates containing ampicillin and chloramphenicolthat selected for both the Lambda ZAP II vector in its plasmidpropagating form and the pBCAR plasmid. Several colonies showeddifferent color tones but only one exhibited a conspicuous brown-redpigment. This colony presumed to contain a xanthophyll biosynthesis genewas selected for further analysis described hereinbelow in the followingExamples.

Example 2 Analysis of the βC-4-oxygenase Activity in Escherichia coli

The red-brown colony presumed to contain a xanthophyll biosynthesis gene(see Example 1 above) was streaked and further analyzed. First, therecombinant ZAP II plasmid carrying the cDNA clone that was responsiblefor xanthophyll synthesis in Escherichia coli was isolated by preparingplasmid DNA from the red-brown colony, transfecting it to Escherichiacoli cells of the strain XL1-Blue and selection on ampicillin-containingmedium. This plasmid, designated pHPK (pHPK is a Lambda ZAP II vectorcontaining an insert isolated from the red-brown colony), was used totransform β-carotene-producing Escherichia coli cells (Escherichia coliSOLR strain that carry the plasmid pBCAR shown in FIG. 4) resulting inthe formation of red-brown colonies. Carotenoids from this transformant,as well as from the host cells (as control) were extracted by acetoneand analyzed by HPLC.

HPLC analysis of carotenoids of the host bacteria which synthesizedβ-carotene (Escherichia coli SOLR strain that carry the plasmid pBCARshown in FIG. 4), as compared with a brown-red colony, revealed thatonly traces of β-carotene were observed in the transformant cells whilea new major peak of canthaxanthin and another minor peak of echinenoneappeared [described in detail by Lotan and Hirschberg (1995) FEBSletters 364: 125-128]. These results indicate that the CDNA in plasmidpHPK, designated crtO encodes an enzyme with β-C-4-oxygenase activity,which converts β-carotene to canthaxanthin via echinenone (see FIG. 14).It is, therefore concluded that a single enzyme catalyzes this two-stepketonization conversion by acting symmetrically on the 4 and 4′ carbonsof the β- and β′-rings of β-carotene, respectively.

Example 3 Production of Astaxanthin in Escherichia coli Cells

To determine whether β-carotene hydroxylase (e.g., a product of the crtZgene of Erwinia herbicola) can convert thus produced canthaxanthin toastaxanthin and/or whether zeaxanthin converted from β-carotene byβ-carotene hydroxylase can be converted by β-C4-oxygenase toastaxanthin, the crtO cDNA of Haematococcus pluvialis thus isolated, wasexpressed in Escherichia coli cells together with the crtZ gene ofErwinia herbicola. For this purpose, Escherichia coli cells of strainSOLR were transfected with either plasmid pASTA alone containing, asshown in FIG. 8, both crtZ and crtO or, alternatively with bothplasmids, pHPK containing, as shown in FIG. 6, crtO, and pZEAXcontaining, as shown in FIG. 5, crtZ. Carotenoids in the resultingtransformed cells were extracted and analyzed by HPLC as describedabove. The results, given in Table 1, show the composition ofcarotenoids extracted from the cells containing the plasmid pASTA.Similar carotenoid composition is found in Escherichia coli cells whichcarry both pHPK and pZEAX.

TABLE 1 Carotenoid % of total carotenoid composition β-Carotene 8.0Echineone 1.7 β-Cryptoxanthin 4.2 Canthaxanthin 4.2 Zeaxanthin 57.8Adonirubin 1.0 Adonixanthin 17.9 Astaxanthin 5.2

The results presented in Table 1, prove that carotenoids possessingeither a β-end group or a 4-keto-β-end group act as substrates for thehydroxylation reactions catalyzed by crtZ gene product at carbons C-3and C-3′. The hydroxylation of β-carotene and canthaxanthin results inthe production of zeaxanthin and astaxanthin, respectively. Thesehydroxylations result in the production of astaxanthin and theintermediate ketocarotenoids, 3-hydroxyechinenone, adonixanthin andadonirubin. These results further demonstrate that astaxanthin can beproduced in heterologous cells by expressing the gene crtO together witha gene that codes for a β-carotene hydroxylase.

Example 4 Sequence Analysis of the Gene for β-carotene C-4-oxygenase

The full length, as was determined by the presence of a poly A tail, ofthe cDNA insert in plasmid pHPK (1771 base pairs) was subjected tonucleotide sequence analysis. This sequence, set forth in SEQ ID NO:1,and its translation to an amino acid sequence set forth in SEQ ID NO:3(329 amino acids), were deposited in EMBL database on May 1, 1995, andobtained the EMBL accession numbers X86782 and X86783, respectively.

An open reading frame (ORF) of 825 nucleotides (nucleotides 166 through1152 in SEQ ID NO:3) was identified in this sequence. This ORF codes forthe enzyme β-carotene C-4-oxygenase having 329 amino acids set forth inSEQ ID NO:4, as proven by its functional expression in Escherichia colicells (see Example 3 above). The gene for this enzyme was designatedcrtO.

Example 5 Transformation of Cyanobacteria with crtO

The plasmid DNA of pPAN3.5-KETO, shown in FIG. 9, was transfected intocells of the cyanobacterium Synechococcus PCC7942 according to themethod described by Golden [Golden SS (1988) Mutagenesis ofcyanobacteria by classical and gene-transfer-based methods. MethodsEnzymol 167: 714-727]. The cyanobacterial cells were plated on BG11medium-containing petri dishes that contained also chloramphenicol.Colonies of chloramphenicol-resistant Synechococcus PCC7942 whichappeared after ten days were analyzed for their carotenoid content. Asdetailed in Table 2 below, HPLC analysis of these cells revealed thatthe major carotenoid components of the cells was β-carotene, echinenone,canthaxanthin, adonirubin and astaxanthin. A similar analysis of thewild type strain and of Synechococcus PCC7942 transfected with a plasmidin which the orientation of the crtO gene is reversed (not shown), whichis therefore not capable of producing an active protein, did notrevealed production of echinenone, canthaxanthin, adonirubin andastaxanthin.

These result prove that crtO of Haematococcus pluvialis can be expressedin cyanobacteria and that its expression provided a β-C-4-oxygenaseenzymatic activity needed for the conversion of β-carotene tocanthaxanthin. This result further demonstrates that the endogenousβ-carotene hydroxylase of Synechococcus PCC7942 is able to convert thusproduced canthaxanthin to astaxanthin. Since the carotenoid biosynthesispathway is similar in all green photosynthetic organism [see FIGS. 1 and10 and, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J(1992) A single polypeptide catalyzing the conversion of phytoene toζ-carotene is transcriptionally regulated during tomato fruit ripening.Proc Natl Acad Sci USA 89: 4962-4966] it is deduced that astaxanthin canbe produced in algae, and higher plants by expressing crtO in any tissuethat express also the endogenous β-carotene hydroxylase. It is furtherdeduced that astaxanthin can be produced by any organism provided itcontains either endogenous or engineered β-carotene biosynthesispathway, by expressing crtO in any tissue that express either endogenousor genetically engineered β-carotene hydroxylase.

TABLE 2 Carotenoid % of total carotenoid composition β-Carotene 31.5Echinenone 18.5 Canthaxanthin 16.1 Zeaxanthin 22.3 Adonirubin 6.0Astaxanthin 5.6

Example 6 Determining the Chirality Configuration of AstaxanthinProduced in Heterologous Systems

The chirality configurations of astaxanthin produced by Escherichia colicells, as described under Example 3 hereinabove, and by cyanobacteriumSynechococcus PCC7942 cells, as described in Example 5 hereinabove, weredetermined by HPLC of the derived diastereoisomeric camphanates of theastaxanthin [Renstrom B, Borch G, Skulberg M and Liaaen-Jensen S (1981)Optical purity of (3S,3S′)-astaxanthin from Haematococcus pluvialis.Phytochem 20: 2561-2565]. The analysis proved that the Escherichia coliand Synechococcus PCC7942 cells described above, synthesize pure(3S,3′S) astaxanthin.

Example 7 Transformation of a Higher Plant with crtO

Producing natural astaxanthin in higher plants has two anticipatedbenefits. First, as a pure chemical, astaxanthin is widely used as feedadditive for fish. It is a potential food colorant suitable for humansconsumption and has potential applications in the cosmetic industry.Second, inducing astaxanthin biosynthesis in vivo in flowers and fruitswill provide attractive pink/red colors which will increase theirappearance and/or nutritious worth.

In flowers and fruits carotenoids are normally synthesized andaccumulated to high concentration in chromoplasts, a typicalpigment-containing plastids, thus providing typical intense colors tothese organs. Inducing synthesis of astaxanthin in chromoplasts enablesthe accumulation of high concentration of this ketocarotenoid.Over-expression of carotenoid biosynthesis genes which results inelevated concentrations of carotenoids in chloroplasts, or otheralterations in carotenoid composition in chloroplasts may damage thethylakoid membranes, impair photosynthesis and thus is deleterious tothe plants. In contrast, increase of carotenoid concentration oralteration in carotenoid composition in chromoplasts do not affect theviability of the plant nor the yield of fruits and flowers.

Thus, gene-transfer technology was used to implant the crtO geneisolated from the alga Haematococcus pluvialis, as described, into ahigher plant, in such a way that its expression is up-regulatedespecially in chromoplast-containing cells.

To this end, a T-DNA containing binary plasmid vector as shown in FIG.12 was assembled in E. coli from the promoter and coding DNA sequencesof the transit peptide encoded by the Pds gene from a tomato speciesLycopersicon esculentum, linked to the coding DNA sequence of crtO fromH. pluvialis. Upon stable transfer of this DNA construct viaAgrobacterium-mediated transformation into a tobacco (Nicotiana tabacumNN) plant to form a transgenic plant, as described under methods above,the plant acquired the ability to produce ketocarotenoids especially inflower tissues (chromoplast-containing cells). It should be noted thatthe Pds gene promoter is capable of directing transcription andtherefore expression especially in chloroplasts and/orchromoplasts-containing tissues of plants. It should be further notedthat the transit peptide encoded by part of the Pds coding sequence iscapable of directing conjugated (i.e., in frame) proteins into plantchromoplasts and/or chloroplasts.

As shown in FIG. 15, in chromoplasts-containing cells, such as in thenectary tissue of the flower of tobacco, this DNA construct inducesaccumulation of astaxanthin and other ketocarotenoids to a higher levelwhich alters the color from the normal yellow to red.

Concentration and composition of carotenoids in chloroplasts-containingtissues, such as leaves, and in chromoplast-containing tissues, such asflowers, were determined in the transgenic plants and compared to normalnon-transformed plants.

Carotenoids compositions in leaves (chloroplasts-containing tissue) andin the nectary tissue of flowers (chromoplast containing tissue) of wildtype and transgenic tobacco plants were determined by thin layerchromatography (TLC) and by high pressure liquid chromatography (HPLC)as described above.

Total carotenoids concentration in leaves (chloroplasts-containingtissue) and in the nectary tissue of flowers (chromoplast containingtissue) of wild type and transgenic tobacco plants are summarized inTables 3 below.

Percents of carotenoids composition in leaves of wild-type andtransgenic tobacco plants are summarized in Tables 4 below.

Percents of carotenoids composition in the nectary tissue of flowers ofwild-type and transgenic tobacco plants are summarized in Tables 5below.

TABLE 3 μg carotenoids per gr fresh weight Wild-type Transgenic withcrtO Leaf (Chloroplasts) 200 240 Nectary tissue (Chromoplasts) 280 360

TABLE 4 % of total carotenoids composition in chloroplasts-containingtissue (leaf) Wild-type Transgenic β-carotene 29.9 26.7 neoxanthin 5.05.9 violaxanthin 11.6 18.1 antheraxanthin 4.9 2.6 lutein 43.9 41.4zeaxanthin 4.7 4.3 astaxanthin + adonirubin 0.0 1.0

TABLE 5 % of total carotenoid composition in chromoplasts-containingtissue (flower) Wild-type Transgenic beta-carotene 58.1 21.0violaxanthin 40.3 1.5 lutein 0.0 1.1 zeaxanthin 1.6 1.0hydroxyechinenone 0.0 13.7 3′hydroxyechinenone 0.0 4.1 adonirubin 0.022.4 adonixanthin 0.0 8.7 astaxanthin 0.0 26.5

Please note the elevated content of hydroxyechinenone,3′hydroxyechinenone, adonirubin, adonixanthin and astaxanthin especiallyin the chromoplast containing tissue of the transgenic tobacco plants.

Thus, the present invention successfully addresses the shortcomings ofthe presently known configurations by enabling a relatively low costbiotechnological production of (3S,3′S) astaxanthin by providing apeptide having a β-C-4-oxygenase activity; a DNA segment coding for thispeptide; an RNA segments coding for this peptide; a recombinant DNAmolecule comprising a vector and the DNA segment; a host containing theabove described recombinant DNA molecule or DNA segment; and of a methodfor biotechnologically producing (3S,3′S) astaxanthin or a food additivecontaining (3S,3′S) astaxanthin, using the host.

While the invention has been described with respect to a limited numberof embodiments, it will be appreciated that many variations,modifications and other applications of the invention may be made.

1. An isolated nucleic acid segment comprising a nucleotide sequenceencoding a polypeptide at least 95% identical to SEQ ID NO: 4, saidpolypeptide having a β-carotene-C-4-oxygenase activity.
 2. A recombinantvector DNA molecule comprising the nucleic acid segment as of claim 1.3. A host comprising a recombinant vector DNA molecule as in claim 2,said host is selected from the group consisting of a microorganism and aplant.
 4. A host comprising the nucleic acid segment of claim 1, saidhost is selected from the group consisting of a microorganism and aplant.
 5. A food additive comprising the host of claim
 3. 6. A foodadditive comprising the host of claim
 4. 7. A transgenic plantexpressing a transgene including a nucleotide sequence encoding apolypeptide at least 95% identical to SEQ ID NO: 4, said polypeptidehaving a β-carotene C-4-oxygenase activity.
 8. A recombinant DNA vectorcomprising a first polynucleotide encoding a polypeptide for directing aprotein into plant chloroplasts or chromoplasts and an in frame secondpolynucleotide encoding a polypeptide at least 95% identical to SEQ IDNO: 4, said polypeptide having β-carotene C-4-oxygenase activity.
 9. Therecombinant DNA vector as in claim 8, wherein said first DNA segment isderived from the Pds gene of tomato.